Original values used to build graphs.

(XLSX)</p

Protein structure simulation of <i>T</i>. <i>gondii</i> MIC3 peptide.

SWISS-MODEL (https://swissmodel.expasy.org/) was used to simulate the tertiary structure of MIC3 peptide. The model validation parameters are as follows: MolProbity Score 2.54, Clash Score 3.81, Ramachandran Favoured 77.78%. (TIF)</p

S1 Fig -

Tlr11 knock-down efficiency in RAW264.7 cells 24 h (A) and 48 h (B) after transfection.Tlr11 expression levels were knocked-down by siRNA targeting Tlr11 (siTlr11). A control siRNA (siCtrl) that targets a scrambled sequence were used as control. 24 h after transfection, RAW264.7 cells were treated with 4 μg/ml MIC3 and OVA for another 24 h. The mRNA levels of Tlr11 and Gapdh were measured 24 h and 48 h after siRNA transfection using qRT-PCR. 1 = no change. Each bar indicates the mean value ± S.D. (n = 3 independent replicates). (TIF)</p

Original images for Western blotting.

(DOCX)</p

Amino acid sequence alignment between <i>T</i>. <i>gondii</i> MIC3 peptide and <i>T</i>. <i>gondii</i> profilin-like protein.

Software Bioedit was used to compare the amino acid sequences of T. gondii MIC3 peptide and profilin-like protein. (TIF)</p

The location of MIC3 in RAW264.7 cells detected by laser confocal microscopy.

To characterize the cellular localization of MIC3, cells were incubated with primary anti-MIC3 mouse monoclonal antibody (Anti-MIC3) and PE-conjugated antibody against mouse F4/80 overnight at 4°C. After being washed, cells were incubated with secondary Alexa Fluor 488 AffiniPure goat anti-mouse IgG antibody (Anti-IgG). DAPI: cell nucleus; F4/80: macrophage surface marker; MIC3: microneme protein 3 of T. gondii.</p

The role of MyD88 in inflammatory immune response induced by MIC3 in RAW264.7 cells.

(A) The levels of TNF-α secreted by RAW264.7 cells after 24 h-treatment of 4 μg/ml MIC3 and OVA. To inhibit the activity of MyD88, RAW264.7 cells in group MIC3+ST2825 were preincubated with 20 μg/ml MyD88 inhibitor ST2825 for 3 h. The secretion of TNF-α was analyzed by ELISA. Each bar indicates the mean value ± S.D. (n = 4 independent replicates). Significance was analyzed using one-way ANOVA. ***, P (B) The mRNA levels of Tnf-α and polarization-related genes after the pretreatment of MyD88 inhibitor ST2825 and 24 h-treatment of MIC3 in RAW264.7 cells. Data are expressed as the means ± S.D. (n = 4 independent replicates). 1 = no change. (C) Ly6C expression in MyD88-inhibited RAW264.7 cells. Ly6C expression in RAW264.7 cells preincubated with 20 μg/ml MyD88 inhibitor ST2825 and treated with 4 μg/ml OVA and 4 μg/ml MIC3 were evaluated by flow cytometry. One representative of three independent experiments is shown. (D) Overlay of representative histograms showing Ly6C expression in MyD88-inhibited RAW264.7 cells treated with 4 μg/ml OVA and 4 μg/ml MIC3. (E) The percentage of Ly6C+ cells in F4/80+CD11b+ gated RAW264.7 cells. Data are expressed as the means ± S.D. (n = 3 independent experiments). Significance was analyzed using one-way ANOVA. ***, P (F) The MFI of Ly6C expression in F4/80+CD11b+ gated RAW264.7 cells. Each bar indicates the mean value ± S.D. (n = 3 independent experiments). Significance was analyzed using one-way ANOVA. ***, P (G) The effects of MyD88 inhibitor ST2825 on MIC3 and LPS-induced NF-κB p65 phosphorylation. The NF-κB p65 phosphorylation of RAW264.7 cells was evaluated by Western blotting.</p

Primers used in this study.

(DOCX)</p

MIC3-induced innate immune responses in THP-1 derived macrophages and cytokine responses in RAW264.7 cells.

(A) The levels of TNF-α secreted by THP-1 derived macrophages after 24 h-treatment of MIC3. THP-1 cells were incubated with PMA to differentiate into THP-1 derived macrophages. Then 4 μg/ml OVA, 4 μg/ml MIC3 and 1 μg/ml LPS were added into the culture medium respectively. The levels of TNF-α were evaluated by ELISA. Data are expressed as the means ± S.D. of six independent samples for each group. Significance was determined by one-way ANOVA. **, P n.s., P > 0.05. (B) The mRNA levels of polarization and inflammation-related genes after 24 h-treatment of MIC3 in THP-1 derived macrophages. Data are expressed as the means ± S.D. of three independent samples for each group. (C) NF-κB p65 phosphorylation of THP-1 derived macrophages after the treatment of MIC3. The NF-κB p65 phosphorylation of THP-1 derived macrophages after 24 h-treatment of 4 μg/ml MIC3, 4 μg/ml OVA and 1 μg/ml LPS was detected by Western blotting. (D) Cytokines secreted by mouse RAW264.7 cells after the treatment with MIC3. The levels of IL-6, IL-10 and IL-12p40 secreted by RAW264.7 cells after 24 h-treatment of 4 μg/ml MIC3 and OVA were detected by ELISA. Data for IL-6 and IL-12p40 are expressed as the means ± S.D. (n = 3 independent experiments). Data for IL-10 are expressed as the means ± S.D. (n = 4 independent experiments). Significance was determined by the two-tailed Student’s t-test. ***, P n.s., P > 0.05. (E) Cytokines secreted by THP-1 derived macrophages after the treatment with MIC3. The levels of IL-6, IL-10 and IL-12p40 secreted by THP-1 derived macrophages after 24 h-treatment of 4 μg/ml MIC3 and OVA were detected by ELISA. Data are expressed as the means ± S.D. (n = 4 independent experiments). Significance was determined by the two-tailed Student’s t-test. ***, P n.s., P > 0.05.</p

The expression levels of inflammation markers in RAW264.7 cells after 24 h-treatment of MIC3.

(A) Ly6C expression in RAW264.7 cells. After 24 h-treatment of 4 μg/ml OVA and 4 μg/ml MIC3, Ly6C expression on F4/80+CD11b+ gated RAW264.7 cells were evaluated by flow cytometry. One representative of three independent experiments is shown. (B) Overlay of representative histograms showing Ly6C expression in RAW264.7 cells treated with 4 μg/ml OVA and 4 μg/ml MIC3. (C) The percentage of Ly6C+ cells in F4/80+CD11b+ gated RAW264.7 cells. Data are expressed as the means ± S.D. (n = 3 independent experiments). Significance was determined by the two-tailed Student’s t-test. ***, P (D) The mean fluorescence intensity (MFI) of Ly6C expression in F4/80+CD11b+ gated RAW264.7 cells. Data are expressed as the means ± S.D. of three independent samples for each group. Significance was determined by the two-tailed Student’s t-test. ***, P (E) The mRNA levels of inflammatory genes after 24 h-treatment of 4 μg/ml MIC3 in RAW264.7 cells. Data are expressed as the means ± S.D. of four independent samples for each group.</p