Key Laboratory of Pathogen Biology of Jiangsu Province, Department of Pathogen Biology, Nanjing Medical University, Nanjing, Jiangsu, China.
PLoS Negl Trop Dis. 2023 Feb 2;17(2):e0011105. doi: 10.1371/journal.pntd.0011105. eCollection 2023 Feb.
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.
刚地弓形虫是全球最成功的寄生虫。了解刚地弓形虫如何在不同宿主中诱导不同的免疫反应是非常重要的。在本研究中，我们发现刚地弓形虫微线蛋白 MIC3 的一个肽段可诱导小鼠巨噬细胞 RAW264.7 细胞产生 TNF-α、NF-κB 磷酸化、iNOS 转录和 Ly6C 表达。MyD88 抑制、针对 TLR11 的小干扰 RNA 和 CRISPR/Cas9 介导的 TLR11 敲除均降低了 MIC3 诱导的 TNF-α 产生、NF-κB 磷酸化、iNOS 转录和 Ly6C 表达。此外，我们使用免疫荧光法确定了 MIC3 肽在小鼠巨噬细胞中的位置。MIC3 既能与小鼠巨噬细胞膜结合，又能进入细胞。这些结果表明，MIC3 通过 TLR11/MyD88/NF-κB 途径触发了小鼠巨噬细胞的免疫反应。已知人类巨噬细胞缺乏 TLR11。我们预测 MIC3 在人类巨噬细胞中诱导的免疫反应与在小鼠巨噬细胞中明显不同。不出所料，MIC3 肽未能诱导人 THP-1 衍生巨噬细胞中 TNF-α 的表达、iNOS 的表达和 NF-κB 的磷酸化。MIC3 通过 TLR11 诱导巨噬细胞免疫反应。有趣的是，MIC3 的氨基酸序列与众所周知的 TLR11 配体原肌球蛋白完全不同，原肌球蛋白可产生强烈的 IL-12p40、TNF-α 和 IL-6 反应。与原肌球蛋白形成鲜明对比的是，MIC3 不能诱导小鼠 RAW264.7 细胞和人 THP-1 衍生巨噬细胞中 IL-12p40 的表达。此外，MIC3 肽的模拟三级结构与原肌球蛋白的晶体结构差异很大，这表明 MIC3 可能是与原肌球蛋白不同的配体。这些关于 MIC3 和 TLR11 的发现将为我们提供有关弓形虫病发病机制和宿主-寄生虫相互作用过程中共同进化的重要见解。
