TRIF Can Be a Suppressor of TLR Signaling Figure 7. rEA-triggered Erk1/2 phosphorylation is MyD88 dependent. C57BL/6 WT or MyD88-KO mice were injected with 100 ng of rEA. Spleen (A) and liver (B) tissues were collected at the indicated time points and processed as described in Materials and Methods. p-Erk1/2 and Erk2 levels were determined by Western blot analysis using LI-COR Odyssey. To control for loading, quantification was performed after normalizing the pErk1/2 to Erk2 levels. Three independent experiments representative of this data are shown. (C) Representative blots: spleen (top), liver (bottom). doi:10.1371/journal.pone.0022064.g007 (Cell Signaling, Inc., Boston, MA) and Erk2 (Santa Cruz Biotechnologies, Santa Cruz, CA). Blots were scanned and bands were quantified using Licor’s Odyssey scanner [41]. For data analysis, the fluorescence of pErk1/2 bands was normalized to Erk2 bands prior to quantification. Mouse Dendritic Cells isolation and mIL12p70 ELISA An in vitro bioassay originally developed to monitor rEA activity during its purification and production, as well as provide some insight into the mechanism of action of rEA, was previously described [8]. The assay is based upon monitoring the release of mIL12(p70) from mouse DCs as an indicator of rEA-induced DC activation. Mouse DCs (CD11c+ splenocytes) were isolated from C57BL/6 WT, MyD88-KO, TRIF-KO, or MyD88/TRIF-DKO mice using the magnetic assisted cell separation (MACS) system (Miltenyi Biotech, Auburn, CA) utilizing the established protocols from the manufacturer and previously described [8]. DC sorting via flow cytometry resulted in .95% pure CD11c positive cell population (Figure S8). The viability of recovered DCs was ,90%, as measured by trypan blue viability staining. Figure 8. TRIF acts as a negative regulator of rEA-induced signaling and downstream responses in DCs: model of action. rEA is a protein derived from Eimeria tenella [8,11] and is highly homologous to Toxoplasma gondii profilin-like protein [14]. T. gondii has been shown to signal, at least in part, through TLR11; therefore, it is likely that TLR11 is one of the main pattern recognition receptors (PRRs) utilized by rEA. We have shown that rEA-triggered responses in vivo are completely dependent on MyD88. MAP kinases consequently become activated downstream of MyD88. Importantly, functional TRIF protein inhibited rEA-mediated signaling. DCs are a major cell type in mediating rEA responses and, under TRIF’s inhibitory effects, have mitigated induction of surface expression of maturation markers and stunted release of pro-inflammatory cytokines/ chemokines. Release of these molecules is critical for rapid amplification of immune responses and is mediated by autocrine and paracrine signaling [16]. We have confirmed that TRIF protein reduces release of pro-inflammatory cytokines/chemokines in response to rEA, resulting in reduced activation of NK, NKT, T, and B cells as well as reduced IFNc production by NK cells. doi:10.1371/journal.pone.0022064.g008 PLoS ONE | www.plosone.org 13 July 2011 | Volume 6 | Issue 7 | e22064