Mechanism Of PBX1-Deficient Decidual NK Cells Inducing Adverse Pregnancy Outcome

Unexplained recurrent spontaneous abortion（URSA）is a pregnancy disease that affects the physical and mental health of women of childbearing age,and its causes are complex and the clinical effectiveness of current treatment options is controversial.The available literature suggests that imbalance of immune tolerance at the maternalfetal interface may be one of the important pathogenic mechanisms of URSA.As the most predominant immune cell at the maternal-fetal interface in early gestation,altered status of decidual natural killer（dNK）cells may have an impact on the maintenance of immune tolerance,which is detrimental to the normal course of pregnancy.Impaired expression of the transcription factor PBX1 in dNK cells is associated with the pathogenesis of URSA.The expression level of PBX1 in dNK cells of patients with recurrent miscarriage is significantly lower than that of healthy women,and the low level of PBX1 limits the secretion of downstream growth-promoting factors（GPFs）,which in turn affects the normal growth and development of the embryo.However,the effects of PBX1 deficiency in vivo go beyond the restriction of GPFs secretion,so we would like to further investigate whether PBX1-dNK cells in URSA patients may induce adverse pregnancy outcomes through other mechanisms.Identifying the mechanisms by which dNK cells induce adverse pregnancy outcomes in patients with URSA can effectively drive the progress of disease treatment.It has been found that dNK cells in women with pregnancy-related disorders（e.g.,pre-eclampsia,gestational diabetes）secrete more inflammatory factors such as TNFa,exhibit embryotoxicity and IL-6.Similarly,higher levels of inflammatory factors were also released from dNK cells in some patients with URSA;therefore,inflammation may be one of the potential mechanisms by which dNK cells induce adverse pregnancy outcomes.This paper focuses on Pbxl^f/f;Ncr1 cre mice as a mouse model of abnormal pregnancy and investigates whether PBX1-NK cells affect the normal pregnancy process through the inflammatory pathway,and the following results were obtained:1.Pbx1^f/f;Ncr1cr mice have abnormal pregnancy outcomeWe used Doppler ultrasound to monitor the pregnancy status of Pbx1^f/f;Ncr1^cre mice and wild-type mice at gd6.5 and gd12.5,we found that the embryo implantation area of Pbx1^f/f;Ncr1^cre mice was smaller.Subsequently,we measured the weight and body length of the fetus at gd16.5,and found that the absorption rate of Pbx1^f/f;Ncr1^cre mouse embryos increased,and the surviving fetus showed significant growth restriction.It was comprehensively detennined that Pbx1f;f;Ncr1^cre mice had adverse pregnancy outcomes.2.PBX1-cells upregulate the expression of LCN2We reanalyzed the transcriptome data of Pbx1^f/f;Ncr1^cre mouse dNK cells in the database and found that dNK-Pbx1CKO cells upregulate the expression of genes related to pathways such as inflammation as well as immunity,and the Lcn2 gene was verified and screened by RT-PCR,and it was determined by flow cytometry that dNK-Pbx1CKO cells also highly express LCN2 protein.3.Inflammatory cells arise at the maternal-fetal interface of Pbx1^f/f;Ncr1Cre miceBy performing flow cytometry testing of decidua as well as spleen tissue from gd11.5 mice,we found increased infiltration of macrophages as well as neutrophils in the maternal-fetal interface of the defective mice.At the same time,the proportion of CD11b+dNK was also significantly increased,and the maternal-fetal interface showed a stronger inflammatory state,and the maternal-fetal interface exhibited a stronger inflammatory state.4.LCN2 causes fetal dysplasia in vivoWe injected recombinant LCN2 protein into mice every 3 days from gd0.5 to simulate the high-level LCN2 environment during pregnancy.By using Doppler ultrasonography to monitor pregnancy,we found that the area of implantation of mouse embryos was reduced and the weight and body length of surviving embryos were significantly lower after LCN2 protein injection,i.e.,excess LCN2 protein caused fetal growth restriction.5.LCN2 promotes inflammation at the maternal-fetal interface in vivo5.Excess LCN2 enhances the inflammatory response at the maternal-fetal interfaceWe examined the ratio of meconium immune cells in mice injected with LCN2 by flow cytometry,and found that neutrophil infiltration at the maternal-fetal interface was increased in the LCN2 group compared with the control group,and the activation level of neutrophils was higher.The inflammatory response at the maternal-fetal interface of mice was more intense after LCN2 injection.In summary,this study found that PBX1-dNK cells upregulate LCN2 expression at the maternal-fetal interface and that excess LCN2 recruits and activates neutrophils,enhancing the inflammatory response at the maternal-fetal interface and ultimately causing embryonic growth restriction,i.e.,dNK cells deficient in the PBX1 gene are capable of triggering adverse pregnancy outcomes via the inflammatory pathway.This study identifies the potential etiology of URSA and provides a new direction for the subsequent treatment of the disease...