T Cell Immune Response Induced By Acute Toxoplasma Gondii Infection In IL-33 Knockout Mice

Objective:In this study,we constructed an acute toxoplasma infection model via peritoneally injected Toxoplasma gondii RH to analyze the polarization effect of macrophages on T cells in IL-33^-/-mice,so as to explore the immune function of IL-33 in mice infected with Toxoplasma gondii.Methods:1.Genotyping:C57BL/6 wild-type mice and IL-33^-/-mice were genotyped by PCR.2.Survival rate determination:10⁴ tachyzoites of Toxoplasma gondii RH strain were injected intraperitoneally into C57BL/6 wild-type and IL-33^-/-mice,1mL/mouse,and mice in the control group were injected intraperitoneally with normal saline to draw the survival curve of the mice.3.Immunohistochemistry:C57BL/6 wild-type and IL-33^-/-mice were injected intraperitoneally with 10⁴ tachyzoites of Toxoplasma gondii RH strain,1 mL/body,and mice in the control group were injected intraperitoneally with normal saline.Liver,spleen,and brain tissues were extracted by HE staining at 48 hours and 96 hours after infection.4.Flow cytometry analysis:IFN-γand CD195 expression of Th1 from spleen of C57BL/6 wild-type and IL-33^-/-mice before and after 48 hours and 96 hours infection by intraperitoneal injection with Toxoplasma gondii.The expression of CD69 and CD195（CCR5）,CD183（CXCR3）and CD186（CXCR6）of T cells after co-culture of macrophages and T cells of spleen,in vitro.5.ELISA:Splenic T cells and peritoneal macrophages were co-cultured in vitro.After T.gondii infection,the level of CCL5,CXCL16 and CXCL9 in the supernatant were detected.6.T cell chemotaxis detection:Macrophages and spleen T cells were prepared and co-cultured in Transwell chambers for 24 hours after Toxoplasma infection,the number of T cells entering the inferior chamber was counted.7.Statistical analysis methods:SPSS 23.0 was used for statistical analysis.Kaplan-Meier method（KM method）was used to compare the difference in survival rates between wild-type and IL-33^-/-mice infected with and not infected with Toxoplasma gondii,depicting mice.For the survival curve,One-way ANOVA（univariate analysis）was used to analyze the data of normal distribution,and the results of flow cytometry analysis were compared.Differences of P<0.05 were statistically significant.Results:1.The mice identified in the experiment were C57BL/6 wild-type and IL-33gene knockout type.2.The survival time of IL-33^-/-mice was longer than that of wild-type mice（P<0.05）.3.The liver tissue of infected mice showed chronic hepatitis.The pathological changes of wild-type mice were more severe than those of IL-33^-/-mice.The spleens and splenic sinuses of the infected mice were disrupted.The pathological changes of spleen wild-type were heavier.Infection of brain tissue showed acute neuronecrosis,and IL-33^-/-mice showed nerve cuff-like changes that were more severe than wild-type pathological changes.4.Wild-type and IL-33^-/-mice were infected with Toxoplasma gondii via abdominal injection.The IFN-γof splenic Th1 cells from IL-33^-/-uninfected mice was higher than that from wild-type uninfected mice（84.33±3.11%vs 52.66±7.60%,P<0.01）.The CD195 of Th1 was higher from IL-33^-/-uninfected group than that in the infected group（194.50±9.52%vs 99.00±3.87%,P<0.01）.5.Splenic T cells were cultured with peritoneal macrophages in vitro with or without infection.The CD69 expression of T cells with IL-33 deficient uninfection was significantly higher than that from wild type T cells without infection（409.85±11.38%vs 312.43±26.33%,P<0.05）.Meanwhile,the CD195 expression of Th1 cells with IL-33 deficient in uninfected group was higher than that from wild-type T cells in uninfected group（180.14±26.53%vs 121.00±16.68%,P<0.05）.The CD186 expression of Th1 cells with wild-type in uninfected group was higher than that from wild-type in infected group（207.00±19.35%vs 101.28±19.01%,P<0.05）.The CD183 expression of Th1 cells with IL-33 deficient in uninfected group was higher than that from wild-type in uninfected group（166.14±14.42%vs114.00±12.60%,P<0.05）.After infection for 24 hours from the supernatant of macrophages and T cells co-cultured in vitro.CCL5 was significantly increased in IL-33 dificient in infection group than that in uninfection group（1377.21±126.29%vs1062.09±192.59%,P<0.05）.CXCL9 was significantly higher in wild-type in infection group than that in IL-33 dificient in infection group（280.66±84.62%vs183.86±63.56%,P<0.05）.CXCL16 was significantly elevated in wild-type in infection group than that in IL-33 dificient in infection group（8210.41±962.21%vs3493.83±359.42%,P<0.01）.6.There were no statistically differences on macrophage chemotactic T cells in uninfected groups between IL-33 deficient and wild type cells,and infected groups with dosage of 10² RH between IL-33 deficient and wild type cells at different time interval.However,in IL-33 dificient infected group with the dosage of 10⁴ RH for 24hours,the number of T cells chemotaxis was obviously higher than that for 12 hours（7.17±5.39%vs 2.43±1.53%,P<0.05）.Conclusion:1.Toxoplasma gondii infection,the survival rate of IL-33^-/-mice was higher than that of wild-type mice,suggesting that IL-33 knockout has a protective effect against Toxoplasma gondii infection in mice.2.In vivo and in vitro T.gondii infection experiments,there was no significant difference in detection of wild-type and IL-33^-/-mice Th1 cell subsets,suggesting that T.gondii infection may not play a key role in anti-infection through Th1 cell subsets,the specific mechanism need to discuss further.