Transdifferentiation From UILC2s And UILC3s To UILC1s Is Involved In The Abnormal Pregnancy Induced By Toxoplasma Gondii Infection

Objective: Innate lymphoid cells(ILCs)are a type of tissue-resident Innate lymphoid cells that play a variety of roles in mucosal inflammation,including defense against pathogens,maintaining epithelial barrier function,inhibiting symbiotic flora,tissue repair,and metabolic regulation.ILCs are important mediators of mucosal immunity,and a large amount of literature has reported that ILC cells have functional plasticity.We found that uterine ILC2 cells and ILC3 cells also exhibited plasticity under Toxoplasma gondii infection.It showed a significant decrease in the expression of cytokines IL-5,IL-13,IL-17 and IL-22 but the expression of IFN-γ,TNF-a,IL-12 and IL-18 was significantly up-regulated.After Toxoplasma gondii infection,ILC2 cells and ILC3 cells aggregated in the inflammatory area,obtained a similar phenotype of ILC1,and showed an immunosuppressive microenvironment that is conducive to maintaining pregnancy.This study aimed to investigate the plasticity of ILC2 and ILC3 in the uterus of Toxoplasma gondii RH strain infection and its effect on adverse pregnancy outcomes.A model of adverse pregnancy outcome in mice infected with Toxoplasma gondii RH strain was established,and the expression of functional molecules of decidual lymphocytes in mice was determined.Our results suggest that infection with Toxoplasma gondii RH strain altered the immunosuppressive microenvironment at the maternal-fetal interface,leading to adverse pregnancy outcomes.Methods: In this study,C57BL/6 mice on the day before the 7:00pm to 2:1 ratio in male and female mouse mated.Early the next day 7:00 observed vaginal plug was defined as gestation 0.5 days.Methods pregnant C57BL/6 mice were randomly divided into two groups equally.The infection group was intraperitoneally injected with 200 of living T.gondii RH strain tachyzoites on the 3.5th day of gestation,and the normal group of mice was injected with physiological saline.All mice were killed on day 9.5 after gestation.The abnormal embryos were identified by their small size,hemorrhagic and necrotic appearance,or complete resorption compared with normal embryos.The adverse pregnancy rate was calculated as the ratio of abnormal embryo numbers to total embryo numbers.Uteri were removed from sacrificed,mated mice at gd9.5.Surrounding fat,blood vessels,ovaries,and cervix were removed from all uteri.And conceptus tissues were then also removed from the uterine wall except virgin uteri.The remaining uterus tissues including myometrium tissue and decidua basalis were minced and digested with RPMI 1640 containing 5 mg/ml collagenase IV 0.5%BSA,10% FCS,and 10 m M HEPES sodium salt.Digested tissues were filtered through sterile nylon gauze to remove undigested tissue and connective tissue.MNCs were purified on a 40%/70% Percoll gradient.Flow cytometry was used to detect the levels of cytokines such as ifn-γ、tnf –a、 Il-5 and Il-17 produced by ILCs,c NK,tr NK and u ILC1 respectively.IL12Rβ2 and IL18 Ra in ILCs were detected by flow cytometry.Total RNA from uterine tissues of pregnant mice in the experimental group and control group was extracted by tissue grinding method.After reverse transcription into c DNA,RNA levels of related cytokines and related receptors in uterine decidual were detected by fluorescence quantitative PCR,such as Ifng,Tnfa Il-5 Il-13 Il-17 Il-12 Il-18 Il-12 r Il-18 r.Placental congestion and inflammatory cell infiltration of mice in the experimental group and control group were observed by H&E staining.Results: After Toxoplasma gondii infection,the embryo absorption rate of the pregnant mice increased significantly,placental congestion was obvious,and inflammatory cell infiltration increased.We examined total ILCs of CD45 positive cells number and percentage were significantly decreased,group 1 ILCs percentage and cells of CD45 positive cells was also decreased,it is important to note that u ILC2,u ILC3 numbers and percentages were decreased,but u ILC1 number and percentage increased.When the expression of cytokines was detected by fluorescence quantitative PCR,Ifna,Tnfa was increased,while Il-5,Il-13 and Il-17 were decreased,Il-12 and Il-18 were increased.The results of flow cytometry were consistent with the fluorescence quantitative PCR,with the increase of Ifng,Tnfa and the decrease of Il-5 Il-17.And the expression of IFN-γ,TNF-a in c NK and u ILC1 and ILCs was detected by flow cytometry,but there was no significant in tr NK.In addition,the expression of IL12Rβ2 and IL18 Ra was increased in both ILCs and group1 ILC cell.Conclusion: After toxoplasma infection,the phenotype of u ILC2 and u ILC3 changes to u ILC1 under the induction of Il-12 and Il-18,which ultimately destroys the immunosuppressant microenvironment at the mother-fetus interface,leading to adverse pregnancy outcome.