Type Ⅱ Innate Lymphocyte (ILC2s) Immunity In Acute Toxoplasma Gondii Infection In IL-33 Knockout Mice

Objective:In this study,acute Toxoplasma gondii infection in IL-33 knockout mice was used as a model to explore the immunomodulatory function of typeⅡintrinsic lymphocytes.To further understand the function of IL-33 and ILC2s and the relationship between them,to provide experimental basis for the immune response mechanism of Toxoplasma gondii infection,and to provide scientific basis for the new treatment scheme of toxoplasmosis and the further application of this model.Methods:1.The basic expression of ILC2s in the lungs of wild type and IL-33^-/-mice was analyzed in vivo:the genotypes of C57BL/6 wild mice and IL-33^-/-mice were identified by PCR,and the proportion and number of ILC2s cells in the lungs of IL-33^-/-mice and wild type mice were analyzed by ILC2s flow antibody surface labeling and flow cytometry.2.The expression of IL-33 in lung,intestine,spleen,liver and kidney of mice with acute Toxoplasma gondii infection at different time points was analyzed by in vivo experiment:the model of acute Toxoplasma gondii infection was established.10⁴ Toxoplasma gondii（1ml）were intraperitoneally injected into C57BL/6wild-type mice and IL-33^-/-mice,and the control mice were intraperitoneally injected with the same amount of complete culture medium The expression of IL-33 was observed by immunohistochemistry;.3.In vivo,the function of ILC2s in acute Toxoplasma gondii infection was studied:flow cytometry was used to detect the proportion and quantity of ILC2s in the lung of mice and wild type of Toxoplasma gondii infection group and 5-day group;the m RNA level of il-2s related transcription factors and cytokines was detected by real time PCR.4.The peritoneal macrophage model of mice infected with Toxoplasma gondii was established in vitro.The phagocytosis rate of peritoneal macrophages was observed by cell climbing film.The mice were pretreated with recombinant IL-33 for 12 hours and then infected with Toxoplasma gondii.The m RNA expression levels of IL-5 and IL-13 related cytokines were detected by real-time PCR.5.Statistical analysis method:SPSS26.0 software was used for statistical analysis,and independent sample t test was used to compare the two groups of samples.After K group independent sample experimental data were tested by normal distribution test and variance homogeneity test,One-way ANOVA,experimental results and data were expressed as mean plus standard SD by single factor analysis of variance（ANOVA）,and a visual chart was attached.It was considered that significant P<0.05was rejected the original hypothesis.The difference was statistically significant.The K independent samples which did not accord with the homogeneity of variance were statistically analyzed by nonparametric test Kruskal-Wallis H test.When the difference was statistically significant,the test results were further compared by Bonferroni method.Z value（test statistics）and P value（significance）were used to compare the differences between the two groups.P<O.O5 had statistical significance,and visual chart was attached.The graphics are drawn and exported by Graph Pad Prism7 software.Results:1.Compared with the control group,the proportion and number of ILC2s cells in the lungs of IL-33^-/-mice were lower（P<0.05）.2.Compared with the uninfected group,the expression of IL-33 and histopathological changes were different in different tissues of mice infected with Toxoplasma gondii for 5 days.3.Compared with the uninfected group,the expression of IL-33 in lung,intestine,liver,spleen and kidney of mice infected with Toxoplasma gondii increased at all time points（P<0.01）4.In the wild-type and IL-33^-/-mice,compared with the uninfected group,the proportion of ILC2s in the lung of the 3-day group infected with Toxoplasma gondii was increased（P<0.05）,and decreased in the 5-day group infected with Toxoplasma gondii（P<0.05）;the proportion of ILC2s in the lung of the 5-day group infected with Toxoplasma gondii was decreased compared with the 3-day group infected with Toxoplasma gondii（P<0.01）.After statistical analysis,there were significant differences between the groups.There was no significant difference in the proportion and number of ILC2s in the lung of IL-33^-/-mice infected with Toxoplasma gondii for 3 days and 5 days compared with the corresponding groups of wild-type mice（P>0.05）.5.In wild mice,the level of IL-5 m RNA decreased（P>0.05）and the level of IL-13 m RNA was higher in the three days group（P>0.05）,but there was no statistical difference（P>0.05）;The expression of IL-5 m RNA was decreased in the 5 days group compared with the non infected group（P<0.05）,and the level of IL-13 m RNA was also decreased,but there was no statistical difference（P>0.05）.In the IL-33^-/-mice,the level of IL-5 m RNA expression was not significantly different in the three days group compared with the uninfected group,and the level of IL-13 m RNA decreased（P<0.01）;The expression of IL-5 and IL-13 m RNA in the 5-day group was lower than that of the uninfected group（P<0.01）.The results of ILC2s related transcription factors showed that the expression level of GATA3m RNA in the three day group was higher in wild mice than in the uninfected group（P<0.01）,and the expression level of GATA3 m RNA in the 5-day group was lower than that in the three days group（P<0.01）;Compared with the uninfected group,the expression level of RORA m RNA in the three days group decreased（P<0.01）,and the expression level of RORA m RNA in the infected group was lower in the 5 days group than that in the 3 days group（P<0.01）.In IL-33^-/-mice,the expression level of GATA3 m RNA in the infected group increased（P<0.01）and the expression level of RORA m RNA decreased（P<0.01）in the three days group;Compared with the three days group,the expression level of GATA3 m RNA decreased（P<0.01）and the expression level of RORA m RNA was lower in the5-day group（P<0.01）.The expression level of transcription factor GATA3m RNA was consistent with the change of proportion and quantity of flow ILC2cells.The expression of IL-5,IL-13 and GATA3 m RNA was not significant in the three day group and 5-day group of T.gondii infection compared with the wild mice.The expression of RORA m RNA in the 3-day group was lower than that in the wild type group（P<0.01）,and the expression of IL-33^-/-mice in the 3-day group was lower than that in the wild type infection group（P<0.01）,but the expression of the group in the 5 days Group was slightly higher,but the difference between the two groups was not statistically significant（P>0.05）.6.In vitro,compared with the control group,the expression levels of IL-5and IL-13 m RNA in wild and IL-33^-/-mice were increased,but there was no significant difference（P>0.05）.The m RNA expression levels of IL-5 and IL-13were significantly increased after pretreatment with rm IL-33（P<0.05）.Compared with IL-33^-/-group,the m RNA expression levels of IL-5 and IL-13 in IL-33^-/-plus rm IL-33 group were significantly higher（P<0.05）.Conclusion:1.The percentage and absolute number of ILC2s in lung of IL-33 deficient mice were lower than that of wild-type mice;2.Toxoplasma gondii infection can promote the expression of IL-33 in lung,intestine,liver,spleen and kidney;3.In vivo and in vitro experiments showed that IL-33 could promote the activation of ILC2s.The absolute number and proportion of ILC2s in the lung of mice with acute Toxoplasma gondii infection changed significantly.The expression of GATA3,a key transcription factor of ILC2s development and function dependence,was consistent with the change of quantity.It suggested that ILC2s may play an immune role in Toxoplasma gondii infection,but it may not play a role through IL-33,The specific mechanism needs further discussion.