The Effect And Mechanism Of IL-17A On Toxoplasma Gondii Infecting 4T1 Cells

As a specialized intracellular parasite of the terminal subphylum,Toxoplasma gondii can infects most warm-blooded animals,and most of them were infected with atypical symptoms due to the suppression of host immune mechanisms,but among immunodeficient hosts,the bulk proliferation of the parasite can cause severe damage to host cells and tissues.Studies have shown that TLA or weak strains of T.gondii have an ideal inhibitory effect on the varieties of tumours.IL-17 A,an important component of the tumour microenvironment,has also been reported it was to be involved in host immunity against T.gondii.In this study,we investigated the effects of IL-17 A on T.gondii invasion and proliferation as well as on the malignant phenotype of tumour cells,and lays the foundation for the study of IL-17A-mediated tumour cell suppression mechanism of T.gondii.Firstly,exogenous and endogenous IL-17 A proteins were obtained by prokaryotic expression and constructing IL-17 A overexpressing 4T1 cell lines through the technique of lentiviral packaging,respectively.In vitro,exogenous IL-17 A protein and T.gondii were co-cultured with 4T1 tumor cells,respectively,to explore the effects of exogenous IL-17 A and T.gondii on the proliferation of 4T1 tumor cells.Meanwhile,the effects of endogenous IL-17 A on invasion,proliferation and plaque formation of T.gondii tachyzoites and malignant phenotype of 4T1 cells were analyzed using stable overexpressed cell lines.In vivo,the effect of IL-17 A on the development of subcutaneous breast transplantation tumor was evaluated through a mouse model,and the effect of IL-17A-mediated T.gondii on the development of mouse subcutaneous breast transplantation tumor was analyzed.The 4T1 cell line and the stable overexpressed cell line were used to construct the mouse mammary gland subcutaneously transplanted tumor model.The tumor model mice were inoculated intraperitoneally with tachyzoite attenuated strains of T.gondii and TLA to induce against T.gondii immunity.The tumor growth data at different stages were measured and the growth curve was plotted.The pathological changes of tumor tissues were observed by staining.Finally,the expression of polyinflammatory cytokines in peripheral blood of mice was analyzed by RT-q PCR to evaluate the anti-tumor immune response mediated by IL-17 A.The results showed that: The IL-17 A gene with the size of 400 bp was successfully cloned and the exogenous IL-17 A protein with the size of 37 k Da was expressed.Stable overexpression cell line p CDH-IL-17A-4T1 was screened by purinomycin after lentivirus packaging.The overexpression efficiency of overexpression cell line IL-17 A was significantly up-regulated at m RNA and protein levels by RT-q PCR and Westen-blot assay(*p<0.05).In vitro experiments: Different concentrations of IL-17 A were positively correlated with the proliferation rate of 4T1 cells by CCK-8 assay,and T.gondii and TLA inhibited the proliferation of 4T1 cells when co-cultured with 4T1 cells,respectively.CCK-8 assay showed that the proliferation rate of stable overexpressed strains was significantly higher than that of the control group.Transwell assay showed that the invasive ability of stable overexpressed strains was significant enhanced,(*p<0.05).When T.gondii and TLA were co-cultured with stable overexpressed cells,the proliferation rate of stable overexpressed cell lines slowed down.The tachyzoite infected cells were observed by IFA and crystal violet staining respectively,and the proportion of infected cells was calculated.The results showed that overexpression of IL-17 A promoted the invasion,proliferation and plaque formation of T.gondii in different degrees(*p<0.05).In vivo experiments: The growth curves of subcutaneous grafted tumors constructed by4T1 cell line and stable overexpression cell line were measured and plotted respectively,which showed that the tumor growth of stable overexpression cell line was slow,suggesting that IL-17 A inhibited the development of tumor,which was significantly different from the control group(*p<0.01).The relative expression of IFN-γ,IL-12,IL-17 and TNF-α in peripheral blood of mice was significantly up-regulated in the stable overexpressed cell tumor group(*p<0.05).By analyzing the growth curve of subcutaneous transplanted tumors in mice infected with T.gondii attenuated strain or TLA,the attenuated strain and TLA showed an inhibitory effect on breast tumors with an average inhibitory rate of 30%-50%,especially over 50% for stable overexpressed cell tumors.These results suggest that IL-17 A mediates the enhancement effect of T.gondii on tumor inhibition.Toxoplasma infection affected the tumor immune response of mice,especially induced changes in the relative expression levels of cytokines such as IL-4,IL-6,IL-10,IL-12,IL-17,IFN-γ and TNF-α in stable overexpressed tumor mice,in which the relative expression levels of IL-12 were most significantly up-regulated(*p<0.01).The results of tumor histopathological examination showed that the vascular abundance of the stable overexpression cell line tumour model was significantly lower.Infection with T.gondii attenuated strain or TLA reduced tumor cell density to varying degrees,while the abundance of tumor vessels was also decreased.In particular,the combined effect of attenuated strains of T.gondii and overexpression of IL-17 A on tumor was significant.In summary,IL-17 A can promote the proliferation and differentiation of tumor cells 4T1 in vitro,and also promote T.g invasion of 4T1 cells,proliferation in 4T1 cells and phagocytic plaque formation.IL-17 A inhibits the tumorigenic effect of 4T1 cells in vivo and inhibits tumor development;IL-17 A can further inhibit tumor development by inducing a cascade immune effect of IL-12 in anti-Toxoplasma immunity.