TRIF Can Be a Suppressor of TLR Signaling Figure 3. TRIF acts as a negative regulator of rEA-induced MyD88-dependent activation of NK, NKT, T, and B cells in vivo. C57BL/6 WT (N = 3–4), MyD88-KO (N = 3), TRIF-KO (N = 3–4), and MyD88/TRIF-DKO (N = 4) mice were injected with 100 ng of rEA. Splenocytes were harvested at 6 hpi, processed, stained for expression of surface markers (intracellular staining was performed for IFNc), and FACS sorted as described in Materials and Methods. All genotype mock-injected mice (N = 2–3) were included in analysis. Separate sets of WT mice were utilized for comparison with each knockout genotype. Activation of NK, NKT, T, and B cells is shown. The bars represent Mean 6 SEM. Statistical analysis was completed using 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.g003 understood. Earlier studies showed that alum and squalene-based emulsion MF59 promote recruitment and increase antigen uptake by APCs, induce cytokine and chemokine secretions, and the expression of adhesion molecules involved in migration of leukocytes [18,19]. More recently it has also been confirmed that alum and squalene based adjuvants may use the NOD-like receptor protein 3 (NLRP3) inflammasome pathway to activate the innate immune system [20,21]. The Eimeria tenella derived protein, was isolated from bovine small intestinal extracts and was shown to have remarkable anti- cancer activity and be a potent stimulator of innate immune responses in various mouse models in vitro and in vivo [8,22,23]. The rEA protein, we believe, might possess all the properties of an ideal immunologic adjuvant, as it can be fairly inexpensive to produce, is extremely stable and can be stored for long periods of time (over 24 month, data not shown) without losing activity. The rEA has been shown to be safe and very well-tolerated in human clinical trials [13]. In mice, rEA augments activation of the innate immune system, presumably by activating PRRs (TLRs and/or possibly others) and thereby increasing adaptive immune responses to co-administered antigens [11,12]. It has been suggested that TLR11 is involved in rEA signaling [9], a notion that is primarily based on high sequence homology (67%) between rEA and T. gondii profilin-like protein, the latter being the only confirmed ligand for TLR11 [14]. In this study we confirmed that administration into mice, or direct exposure of immune cells to rEA protein results in (1) activation of important immune cell types (DCs, macrophages, NK, NKT, B and T cells), including (2) pro-inflammatory cytokines/chemokines release, both globally and specifically by DCs, and highly robust IFNc production by NK cells, and (3) Erk1/2 phosphorylation. Therefore, we showed that rEA, similarly to other TLR-agonist-based adjuvants, activated an innate immune profile that resulted in robust activation of innate immune cells and induced multiple cytokines/chemokine path- ways. Our results also confirmed that these responses are completely dependent upon MyD88, as genetic knockout of this adaptor protein results in complete ablation of these responses both in vitro and in vivo. When we similarly investigated the role of TRIF, the other major TLR adaptor protein, we encountered unexpected results. Specifically, TRIF-KO mice showed dramatic increases, in immune cell activation and other rEA triggered responses, when compared to rEA treated WT mice. Specifically, we found that IL6, IL12p40, IL2, IL1a, IL1b, and MIP1a production was induced by rEA treatment in TRIF-KO mice in vivo and in DCs derived from these mice in vitro to significantly higher levels as compared to similar assays performed in rEA-treated WT mice. Note, that rEA mediated induction of IL12p70, a cytokine abundantly produced by DCs that activates NK cells, was also found to be negatively regulated by TRIF after exposure to rEA [16,24]. Therefore, in response to rEA-mediated stimulation, TRIF acts as a suppressor of the rEA-induced (MyD88-dependent) activation of DCs, macrophages, NK, NKT, T, and B cells in vivo. Despite the lack of TRIF activity in MyD88/TRIF-DKO mice, rEA responses were still ablated which indicates that in the absence of MyD88, TRIF cannot carry out its suppressive activity. Since the lack of TRIF protein does not rescue the phenotype in MyD88/ TRIF-DKO mice, this confirms an essential role of MyD88 in mediating these responses and suggests that TRIF protein acts as a suppressor downstream of MyD88 and/or MyD88 and TRIF adaptor molecules orchestrating the induction of pro-inflammato- ry immune responses following rEA injection. Numerous studies have described important roles for TRIF as a TLR system adaptor protein that acts to enhance TLR-based signaling (predominantly when TLR3 and TLR4 ligands are interrogated, thereby promoting antimicrobial responses [6,17,25,26]. Specifically, TRIF-KO mice showed dramatically impaired lung clearance of Pseudomonas aeruginosa infections; a response that is correlated with blunted cytokine induction (e.g. RANTES, IL1b, MIP2) and reduced NFkB activation present in both alveolar and peritoneal macrophages from these mice [27]. Lack of TRIF protein also results in reduced induction of antigen specific humoral and cellular immune responses in other models [28]. Specifically, T cells derived from TRIF-KO mice had dramatically reduced IFNc production and CXCR3 expression upon antigen/LPS treatment as compared to WT mice [29]. In DCs, TRIF functionality was confirmed to be important for upregulation of CD40 and CD86 co-stimulatory molecules [30]. Moreover, TRIF protein along with IPS1 (RIGI/Mda5 pathway protein) are key adaptors in mediating PolyIC-triggered adjuvant effects [28]. Conversely, there is very little data available that describes suppressive roles of TRIF protein on TLR/PRR signaling. Specifically, we have previously demonstrated that a lack of functional TRIF protein results in increased transgene (e.g. b-Gal) specific IgG titers in mice injected with Ad5-LacZ, suggesting that TRIF may act as a negative regulator of Ad-mediated antibody responses in mice [31]. Other researchers have demonstrated that TRIF has an inhibitory role in TLR5-mediated responses through induction of TLR5 degradation [32]. This phenomenon may not be limited to TLR5, as it has been suggested that TRIF can also induce degradation of other TLRs, including TLRs 3, 6, 7, 8, 9, and 10 [32]. To determine if the suppressive role of TRIF in DCs is more global than previously considered, we have stimulated isolated DCs with TLR4 (LPS), TLR7/8 (R848) or TLR9 (ODN2006) agonists and found that the presence of the TRIF adaptor protein significantly suppresses release of pleiotropic pro- inflammatory cytokines by DCs in response to these agonists. These studies suggest that suppressive activities of TRIF, in regard to pathogen-induced innate immune responses, may be more prevalent than currently appreciated. Activation of pro-inflammatory cytokines/chemokines is a critical step during DC activation/maturation. It has been suggested that TRIF may be required for inducing immunological tolerance by augmenting IL10 production [33,34]. We found, however, that in rEA (or other TLR agonist) treated TRIF-KO mice, amounts of IL10 (both in plasma and in isolated DCs) were indistinguishable to identically (rEA) treated WT mice. To successfully bridge DC activation to induction of substantial adaptive immune responses (e.g. T cell activation), it is essential to have induction of co-stimulatory molecules such as CD40, CD80, and PLoS ONE | www.plosone.org 7 July 2011 | Volume 6 | Issue 7 | e22064