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

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-alpha production, NF-kappa B 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-alpha production, NF-kappa B 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 trigger the immune responses in mouse macrophages via TLR11/MyD88/NF-kappa B 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-alpha expression, iNOS expression and NF-kappa B phosphorylation in human THP-1 derived macrophages. MIC3 induces 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-alpha 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. Author summaryTo be the most successful parasite worldwide, Toxoplasma gondii develops fascinating ways to coexist with its host and fulfil its life cycle. After infection with T. gondii, the survival time of mice is short, while human beings can coexist with T. gondii for lifetime. Remarkably, in this study we found that a short peptide from T. gondii microneme protein MIC3, which was known to be the circulating antigen after T. gondii infection, was sufficient to trigger the TLR11/MyD88/NF-kappa B pathway and induced macrophage TNF-alpha production, NF-kappa B phosphorylation, iNOS transcription and Ly6C expression in mouse macrophages. Human macrophages lacking TLR11 failed to induce such inflammatory reaction in response to MIC3. Therefore, we speculate that the TLR11-driven deleterious inflammatory immune responses impede the long-term coexistence of host and T. gondii, which might provide the evolutionary pressures for TLR11 expression. Additionally, the amino acid sequence, simulated tertiary structure and triggered immune responses of MIC3 are completely different from the well-known TLR11 ligand profilin. All together, these findings propose MIC3 as a potential different ligand from profilin, and what we learn about MIC3 and TLR11 provides important insights into the evolutionary roles of T. gondii and other TLR11 sensing pathogens.