Identification And Functional Analysis Of Uterine Innate Lymphoid Cells In Mouse Pregnancy

Objective:Innate lymphoid cells(ILCs)were identified as newly natural immune cells.ILCs include three groups for their transcriptional factors and cytokines.NK(natural killer)cells belong to the group 1 ILCs.In recent years,the characteristics and function of uterine NK cells in reproduction has been identified,NK cells are critical to controlling trophoblast invasion,immune tolerance,vascular remodeling and fetal-placental growth.Recently,diverse innate lymphoid cells(ILCs)were identified in human and mouse uterus,but theroles of other than uterine innate lymphoid cells(u ILCs)during pregnancy is unclear.In this study,we identified the characters and function of u ILCs by detecting the percentage and absolutely number of u ILCs,level of cytokines,the influence to pregnancy outcome after ILCs depletion and to explore the function of ILCs in abnormal pregnancy of Toxoplasma gondii infection.Methods: In this study,C57 mice on the day before the 7:00pm to 3:2 ratio in male and female mouse mated.Early the next day 7:00 observed vaginal plug was defined as gestation 0.5 days,the 6.5,9.5 and 13.5 days of pregnancy is defined as early,middle and late gestation,in respectively;separated mononuclear cells in mouse uterus during pregnancy by collagenase IV digestion and fluorescent antibody staining to determine the proportion and the absolute number of u ILCs;liquid nitrogen grinding method for extraction of total m RNA of mice uterus during pregnancy,reverse transcription to c DNAand to detect the level of m RNA expression by fluorescent quantitative PCR;uterine mononuclear cells isolated in vitro cultured with PMA,cells stained to detecttheexpression of IL-5,IFN-?,IL-13,IL-17 and IL-22 fromtotal u ILCs;the distribution of u ILCs in Rag1-/-mouse uterus were located by Immunohistochemical(IHC);flow cytometry to test elimination of u ILCsby antibody in Rag1-/-pregnant mice;to detect the size of uterine spiral artery by H&E in gestation days 10.5,as well as the growth of fetal and placental in gestation days 16.5 after ILCs depletion;The changes of ILCs during abnormal pregnancy were observed after the Toxoplasma gondii infected.Results:The number of u ILCs markedly increased early in gestation,compared with virgin mice,the percentage of group1 ILCs(ILC1s)increased at gd6.5,the absolute number of total group 1 ILCs increased at both gd6.5 and gd13.5.We then examined changes throughout the entire gestation in three subsets of group 1 ILCs,the absolute numbers of all three subsets of group 1 ILCs increased during gestation,especially at late gestation;The absolute number of group 2 ILCs(ILC2s)markedly increased at all stages of pregnancy,peaking at gd6.5,and was the largest subset in number of u ILCs.In addition,the percentage of IFN-?,IL-5 and IL-13 among ILCs increased compared to virgin mice in early gestation.However,the production of IL-17 and IL-22 by u ILCs was not enhanced during pregnancy.Thy1.2+ u ILCs were mainly distributed in the mesometrial lymphoid aggregate of pregnancy(MLAp)and decidua basalis(DB)in middle gestation by IHC.Next,we assessed the morphological aspects of the implantation sites in Rag1-/-females and found that the impaired uterine spiral artery remodeling.At the same time,we initially found that the rate of inflammatory factors IFN-? and TNF-? in the abortion model caused by Toxoplasma gondii infection was significantly higher.Conclusion:These results support the conclusion that u ILCs play important roles in successful pregnancy in mice.