Toxoplasma gondii microneme protein MIC3 induces macrophage TNF-α production and Ly6C expression via TLR11/MyD88 pathway
Fig 6
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 < 0.01. 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 < 0.001. 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 < 0.001. n.s., P > 0.05.