TRIF Can Be a Suppressor of TLR Signaling experiments is shown. The bars represent Mean 6 SD. Statistical analysis was completed using a two-way ANOVA with a Bonferroni post-hoc test (genotypes6rEA treatments). *, ** - Indicate values that are statistically different from those in unstimulated DCs (for the same genotype), p,0.05, p,0.001 respectively. #, ## - Indicate values statistically different from those in WT DCs (for the same rEA dose), p,0.05, p,0.01 respectively. No significant differences between mock-injected animals of different genotypes were detected. No significant activation of IL12p70 was observed in MyD88-KO and MyD88/TRIF-DKO DCs. (B) DC culture media was collected at 18 hours post-rEA stimulation (0.2 ng/ml) and was analyzed for cytokines/chemokines levels using a multiplexed bead array based quantitative system. Statistical analysis was completed using a one-way ANOVA with a Student-Newman-Keuls post-hoc test. The bars represent Mean 6 SD. *, ** - Indicate 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 pro-inflammatory cytokines was observed in MyD88-KO and MyD88/TRIF-DKO animals (only anti-inflammatory IL10 cytokine was induced in MyD88-KO mice). doi:10.1371/journal.pone.0022064.g005 In conclusion, our murine models have shown that rEA activates multiple immune cell types, stimulates pro-inflammatory cytokine/chemokine release, and triggers the MAPK pathway in a MyD88 dependent manner. We have also confirmed that DCs are the main subset of innate immune cells that mediate rEA-triggered responses, thus justifying future studies on isolated DCs and the potential use of rEA adjuvant in a DC-vaccine setting [36]. Importantly, our studies of rEA unveiled a suppressive activity to the TRIF adaptor protein. This clearly justifies future testing of specific TRIF inhibitors or knockdown models prior to rEA/ antigen administration as a means to further enhance induction of antigen specific adaptive immune responses. Whether TRIF acts to negatively regulate other adjuvants or TLR-mediated activa- tions is a question that will require future investigations. The latter, however, highlights the complexities of TLR adaptor functions, and should temper efforts that target these proteins with agonists or antagonists, as the end result of such interventions may run counter to hoped for outcomes, and could be detrimental in some situations [37]. Materials and Methods Animal Procedures All animal procedures were reviewed and approved by the Michigan State University ORCBS and IACUC. Care for mice was provided in accordance with PHS and AAALAC standards (ID number: A3955-01). Adult male C57BL/6 mice were purchased from The Jackson Laboratory (Bar Harbor, ME). MyD88-KO and TRIF-KO mice were kindly provided by Dr. Shizuo Akira. MyD88/TRIF-DKO mice were bred at Michigan State University. Intraperitoneal (IP) injection of animals (2–4 months in age) consisted of 100 ml phosphate-buffered saline solution (PBS, pH 7.4) containing 100 ng rEA protein from Eimeria tenella as previously described [8,11]. rEA protein purification was performed as previously described [8] with minor modifications described [11]. Plasma and tissue samples were obtained and processed at the indicated times post-injection as previously described [11]. Importantly, IP route of rEA admin- istration was confirmed to be more efficient that other widely used routes for adjuvant injection, such as intranasal or subcutaneous [7,8]. To study rEA-triggered activation of immune cells by flow cytometry and bead array methods, plasma and spleen tissues were harvested at 6 hours post injection (hpi), whereas for studies measuring activation of signaling pathways the spleen and liver tissues were harvested at 0, 20, 40, 60, 90, and 120 minutes post- rEA injection. Cytokine and chemokine analysis A mouse 23-plex multiplex based assay was used to determine the indicated cytokine/chemokine concentrations in the plasma collected in vivo or the media collected from cultured DCs per the manufacturer’s instructions (Bio-Rad, Hercules, CA) via Luminex 100 technology (Luminex, Austin, TX) as previously described [38]. Isolation of Splenocytes Splenocytes from individual mice were harvested and processed by physically disrupting and facilitating passage of splenic tissue through a 40 mm sieve, followed by induction of RBC lysis by using 2 ml of ACK lysis buffer (Invitrogen, Carlsbad, CA) per homogenized spleen. Splenocytes were subsequently washed two times with RPMI medium 1640 (Invitrogen, Carlsbad, CA) supplemented with 10% FBS, 2 mM L-glutamine, and 1% PSF (penicillin, streptomycin, fungizone), then resuspended and counted. Cell staining and flow cytometry Splenocyte preparations were evaluated for the presence of DC, macrophages, NK, NKT, T, and B cell activation/maturation markers as previously described [11,39]. 16106 cells were stained with combinations of the following antibodies: APC-CD3, PerCpCy5.5-CD19, PE-Cy7-NK1.1, and PE-CD69 (all 4 mg/ml) or PECy7-Cd11c, APCCy7-CD11b, APC-CD80, Pacific Blue- CD86, Alexa Fluor700-MHCII, FITC-CD40, and PerCpCy5.5- CD3/CD19/NK1.1 (dump channel) (all 4 mg/ml) (BD Bioscienc- es, San Diego, CA). Cells were incubated on ice with the appropriate antibodies in 2.4G2 hybridoma cell supernatant for 30 minutes, then washed and sorted using a BD LSR II instrument. For intracellular staining, 26106 splenocytes were incubated with Brefeldin A (1 mg/ml) in DMEM with 10%FBS/ 16PSF for 4 hours at 37uC. Following incubation, splenocytes were washed two times with FACS buffer, incubated for 15 minutes with purified rat anti-mouse CD16/CD32 Fcc block (BD Biosciences, San Diego, CA), surface stained with CD3-APC and NK1.1-PECy7 (8 mg/ml) for 30 minutes at 4u C, washed with FACS buffer, fixed with 2% formaldehyde (Polysciences, War- rington, PA) for 20 minutes on ice, permeabilized with 0.5% Saponin (Sigma-Aldrich, St. Louis, MO) for 20 minutes at room temperature, and incubated on ice with IFNc-FITC (8 mg/ml) for 2 hours. Samples were analyzed on a BD LSR II instrument using FlowJo software (Tree Star, San Carlos, CA, USA). Western blotting Spleen and liver tissues were homogenized in lysis buffer (20 mM Tris-HCl, pH 7.4, 1 mM EDTA, 150 mM NaCl) containing 1% Triton X-100 with protease inhibitors. Homoge- nized tissues were then centrifuged at maximum speed (13,0006G) for 10 min at 4uC, after which the protein concentration of the supernatant determined using BCA method. Western blotting for pErk1/2 and Erk2 was performed as previously described [40,41,42]. Equivalent concentrations of protein samples were run on polyacrylamide gels and transferred onto nitrocellulose membranes. Blots were then probed with fluorescent antibodies as previously described, which included antibodies against pErk1/2 PLoS ONE | www.plosone.org 10 July 2011 | Volume 6 | Issue 7 | e22064