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. 2023 Feb 2;17(2):e0011105.
doi: 10.1371/journal.pntd.0011105. eCollection 2023 Feb.

Toxoplasma gondii microneme protein MIC3 induces macrophage TNF-α production and Ly6C expression via TLR11/MyD88 pathway

Affiliations

Toxoplasma gondii microneme protein MIC3 induces macrophage TNF-α production and Ly6C expression via TLR11/MyD88 pathway

Jingfan Qiu et al. PLoS Negl Trop Dis. .

Abstract

Toxoplasma gondii is the most successful parasite worldwide. It is of great interest to understand how T. gondii induce different immune responses in different hosts. In this study, we found that a peptide of T. gondii microneme protein MIC3 induced TNF-α production, NF-κB phosphorylation, iNOS transcription and Ly6C expression in mouse macrophage RAW264.7 cells. MyD88 inhibition, small interfering RNA against Tlr11 and CRISPR/Cas9-mediated knock-out of Tlr11 all reduced MIC3-induced TNF-α production, NF-κB phosphorylation, iNOS transcription and Ly6C expression. Additionally, we determined the location of MIC3 peptide in mouse macrophages using immunofluorescence. MIC3 could both adhere to the cell membrane of mouse macrophages and enter the cells. These results suggest that MIC3 triggered the immune responses in mouse macrophages via TLR11/MyD88/NF-κB pathway. It is known that human macrophages lacking TLR11. We predicted that the immune responses induced by MIC3 in human macrophages were significantly different from those in mouse macrophages. As expected, MIC3 peptide failed to induce TNF-α expression, iNOS expression and NF-κB phosphorylation in human THP-1 derived macrophages. MIC3 induced macrophage immune responses via TLR11. Intriguingly, the amino acid sequence of MIC3 is completely different from the well-known TLR11 ligand profilin, which generates a potent IL-12p40, TNF-α and IL-6 response. In marked contrast to profilin, MIC3 could not induce IL-12p40 expression in both mouse RAW264.7 cells and human THP-1 derived macrophages. Furthermore, the simulated tertiary structure of MIC3 peptide shows poor similarity with the crystal structure of profilin, suggesting that MIC3 might be a different ligand from profilin. These findings about MIC3 and TLR11 will provide us with important insights into the pathogenesis of toxoplasmosis and coevolution during host-parasite interaction.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. MIC3 induced TNF-α expression and NF-κB p65 phosphorylation 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. Data are expressed as the means ± S.D. of four independent samples for each group. Significance was determined by the two-tailed Student’s t-test. ***, P<0.001. (B) NF-κB p65 phosphorylation of macrophages after the treatment of MIC3. The NF-κB p65 phosphorylation of RAW264.7 cells after 24 h-treatment of 4 μg/ml MIC3, 4 μg/ml OVA and 1 μg/ml LPS was detected by Western blotting. (C) The NF-κB p65 phosphorylation of RAW264.7 cells after 24 h-treatment of 4 μg/ml MIC3 and 4 μg/ml OVA was evaluated by immunofluorescence assay.
Fig 2
Fig 2. 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 < 0.001. (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 < 0.001. (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.
Fig 3
Fig 3. 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 < 0.001. (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 < 0.001. (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 < 0.001. (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.
Fig 4
Fig 4. 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.
Fig 5
Fig 5. The effects of Tlr11 knock-down and knock-out on MIC3-induced immune responses in RAW264.7 cells.
(A) The effects of Tlr11 knock-down and knock-out on TNF-α secretion of RAW264.7 cells after 24 h-treatment of 4 μg/ml MIC3 or OVA. To knock-down the Tlr11, siRNA targeting Tlr11 (siTlr11) and a control siRNA (siCtrl) were used. CRISPR/Cas9 technology was used to create a Tlr11 knock-out RAW264.7 cell line (Tlr11-/-). Each bar indicates the mean value ± S.D. (n = 3 independent experiments). Significance was analyzed using one-way ANOVA. ***, P < 0.001. n.s., P > 0.05. (B) The effects of Tlr11 knock-down and knock-out on the mRNA levels of polarization-related genes (iNOS and Arg-1) after 24 h-treatment of MIC3 in RAW264.7 cells. Data are expressed as the means ± S.D. (n = 3 independent experiments). 1 = no change. (C) Ly6C expression in Tlr11 knock-down and knock-out RAW264.7 cells. Ly6C expression after 24 h-treatment with 4 μg/ml OVA and 4 μg/ml MIC3 were evaluated by flow cytometry. One representative of three independent experiments is shown. (D) The percentage of Ly6C+ cells 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 < 0.001. **, P < 0.01. n.s., P > 0.05. (E) The MFI of Ly6C expression in F4/80+CD11b+ gated RAW264.7 cells. Data are expressed as the means ± S.D. (n = 3 independent experiments). (F) The effects of Tlr11 knock-down and knock-out on MIC3-induced NF-κB p65 phosphorylation. The NF-κB p65 phosphorylation of RAW264.7 cells was evaluated by Western blotting.
Fig 6
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.

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