Isolated CD11c+ cells were seeded at a density of 0.36105 cells per well into 96-well plates in 200 ml/well complete medium. The complete medium consisted of DMEM/F12 supplemented with 10% FCS, gentamicin (10 mg/ml), recombinant mouse GMCSF (1 ng/ml), recombinant mouse IL4 (1 ng/ml), recombinant mouse IFNc (3 ng/ml), and an agonistic anti mouse CD40 antibody (0.5 mg/ml) (R&D Systems, Minneapolis, MN). This synergistic combination of cytokines has no significant effect on mouse IL12 release, but dramatically enhances the inducing effects of rEA on mouse IL12 release [8]. Mouse DCs were stimulated with rEA (1026–101 ng/ml) overnight (,18 hours) at 37u C in 5% CO2 and 95% ambient air. To study the specificity of TRIF inhibitory effects in DCs, various toll-like receptor agonists were added to mouse DCs, using the following concentrations: rEA (Barros Research Institute, Holt, MI, 100 ng/ml); E. coli 0111.B4 LPS (20 mg/ml); R848 (0.5 mg/ml) and ODN2006 (2.5 mM). LPS, R848 and ODN2006 (TLR4, TLR7/8 and TLR9 agonists respectively) were purchased from InvivoGen, San Diego, CA, reconstituted with endotoxin-free water and diluted in culture media (DMEM/F12+10% FCS). Following incubation, culture medium was analyzed for mouse IL12p70 levels using an ELISA kit and following its enclosed instructions (R&D Systems, Minneapolis, MN) or for 23 mouse cytokines/chemokines using multiplex system (Bio-Rad, Hercules, CA). Statistical analysis Statistically significant differences in immune cells activation assays were determined using two-tailed homoscedastic Student’s t-tests to compare rEA mediated responses in WT mice against each individual knockout genotype in independent experiments (p value,0.05). Statistically significant differences in plasma or media cytokine levels were determined using a one-way ANOVA with a Student-Newman-Keuls post-hoc test (p value,0.05). Furthermore, a two-way ANOVA with a Bonferroni post-hoc test was used to analyze the levels of IL12p70 in media from cultured DCs (genotype6rEA treatments) that were derived from WT, MyD88- KO, TRIF-KO or MyD88/TRIF-DKO mice and treated with several escalating concentrations of rEA. Graphs in this paper are presented as mean of the average 6 SEM, unless otherwise specified. GraphPad Prism software was utilized for statistical analysis. Supporting Information Figure S1 TRIF acts as a negative regulator of rEA- induced MyD88-dependent activation of dendritic cells in vivo. Representative histograms are illustrated on this figure. (TIF) Figure S2 Sorting strategy for CD11c+ cells. Representa- tive figure of the gating strategy for sorting CD11c+ DCs. (A) Splenocytes were stained with CD11c-PECy7 and CD11b-APC- Cy7 antibodies and data were generated using LSR-II cytometer. (B) Splenocytes were stained with CD11c-PECy7 and PerCPCy5.5 conjugated antibodies for CD3, NK1.1, and CD19 to exclude T-, NK-, and B-cells from the analysis. (TIF) Figure S3 TRIF acts as a negative regulator of rEA- induced MyD88-dependent activation of macrophages 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, and FACS sorted as described in Materials and Methods. All genotype mock- injected mice (N = 2–3) were included in analysis. One of two TRIF Can Be a Suppressor of TLR Signaling representative experiments is shown. Separate sets of WT mice were utilized for comparison with each knockout genotype. Activation of CD11b+, CD192, and CD32 macrophages is shown. The bars represent Mean 6 SEM. Statistical analysis was completed using a two tailed homoscedastic Student’s t-tests. *, ** - Indicate values significantly higher (#, ## - lower) from those in mock injected animals (of the same genotype), p,0.05, p,0.001 respectively. (TIF) Figure S4 TRIF acts as a negative regulator of rEA- induced MyD88-dependent activation of macrophages cells in vivo. C57BL/6 WT (N = 3), MyD88-KO (N = 3), TRIF- KO (N = 3), 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, 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. 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 significantly higher (#, ## - lower) from those in mock-injected animals (of the same genotype), p,0.05, p,0.001 respectively. (TIF) Figure S5 TRIF acts as a negative regulator of rEA- induced MyD88-dependent activation of macrophages in vivo. Representative histograms are illustrated on this figure. (TIF) Figure S6 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), MyD88-KO (N = 3), TRIF-KO (N = 3), 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. 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 significantly higher (#, ## - lower) from those in mock-injected animals (of the same genotype), p,0.05, p,0.001 respectively. (TIF) Figure S7 TRIF acts as a negative regulator of rEA- induced MyD88-dependent activation of NK, NKT, T, and B cells in vivo. Representative histograms and plots are illustrated on this figure. (TIF) Figure S8 Validation of CD11c+ DCs isolation. DC sorting resulted in .95% pure CD11c positive cell population. The viability of recovered DCs was ,90% as measured by trypan blue viability staining. (TIF) Acknowledgments We wish to thank the Michigan State University Laboratory Animal support facility for their assistance in the humane care and maintenance of the animals utilized in this work. PLoS ONE | www.plosone.org 14 July 2011 | Volume 6 | Issue 7 | e22064