The Regulatory Mechanism Of LAIRs And Collagen On The Decidual NK Cells In Human First Trimester

Pregnancy is a special immunological event. There is a special immune tolerance microenvironment in the maternal-fetal interface. The crosstalk between different kinds of cells and molecules in the maternal-fetal interface is the key of maternal-fetal immune regulation research. To explain the interaction among the trophoblasts, maternal decidual stromal cells and immune cells is the important for the research of mechanisms in maternal-fetal immune tolerance during pregnancy. It is of great value not only in the reproductive immunology, but also in the tumor and transplantation immunology.LAIR-1 belongs to the inhibitory receptors family, which are involved in controlling the immune balance to prevent improper activation. Upon interaction with their ligands-collagen, LAIR-1 can attenuate the signals provided by activatory receptors, thereby increasing the threshold for activation. The maternal-fetal interface is composed of trophoblasts and maternal decidual cells. More than 70% of decidual immune cells (DICs) are CD56bright CD16-decidual NK which play a very important role in the maternal-fetal immune tolerance.Therefore, our research firstly analyzed the expression of LAIRs and collagens in the maternal-fetal interface and its correlation with abortion. Then we targeted the NK cells in human first trimester. By co-cultured with the trophoblast cells and/or decidual stromal cells in different treatment groups, we illustrated the regulatory roles of the interaction between collagens and LAIRs in the function of decidual NK cells and the intracellular signal transduction pathway.Part I Expression of LAIRs and Collagen and Their Correlation with AbortionObjectives To comparably analyze the expression of LAIRs and collagen in the villi and deciduas tissues from normal pregnancy and abortion. To comparably analyze the collagens expressed in the culture of trophoblast and decidual stromal cells (DSC) and LAIR-1 in NK cells as well.Methods The first-trimester human abortion tissues were collected and trophoblast cells and DSCs were isolated and identified as our previous study. Masson and IH technology were used to analyze the expression of LAIR-1 and collagens in the tissues. PCR and ELISA were used to detect the expression of LAIRs and collagen expressed in the in vitro culture system; FCM was applied to analyze the LAIR-1 expression in the decidual NK cells.Results Masson and IH analysis showed a higher expression of collagens in the tissues from normal pregnancy than that of abortion. The mRNA and protein expression level of collagens in the normal trophoblast and DSCs was also higher than that of abortion. The LAIR-1 expression in the normal pregnancy tissue was higher than that of abortion and the same with the LAIR-1 expression in decidual NK cells.Conclusions There is a correlation between the low expression of collagens in the maternal-fetal interface and abortion. There is also a correlation between the low expression of LAIR-1 in dNK cells and abortion.Part II Effect of the Interaction between LAIR-1 and Collagen on the NK cellsObjectives To analyze the character of the interaction between LAIR-1 and collagen; to analyze the regulation of this interaction on the NK cells.Methods We isolated peripheral NK cells from normal pregnancy woman’s peripheral blood and isolated decidual NK cells from normal early pregnancy deciduas. The purified NK cells were cultured in vitro with collagens in different concentration. The induced expression of LAIR-1 was detected by PCR and FCM. The purified NK cells were also co-cultured with collagens and/or LAIR-2. The activating and inhibitory receptors as well as the Thl/Th2 cytokines and perforin were analyzed by FCM.Results The mRNA and protein expression of LAIR-1 can be induced by collagens in the dose dependent manner. The expression of NKp30 was decreased and the KIR2DL1 was increased in the NK cells after the collagens treatment. The expression of Th1 type cytokines IFN-y and TNF-a was decreased after the treatment. There is no change in the expression of Th2 type cytokines IL-4 and IL-10 after treatment. The expression of perforin was down regulated by collagens and showed a negative correlation with the LAIR-1 expression MFI. The effect of the collagen can be abrogated by the LAIR-2 treatment.Conclusions Collagen alone treatment can induce the expression of LAIR-1 in NK cells and the treatment can down regulate the expression of Thl cytokines and perforin and induce the immune tolerance phenotype in NK cells.Part III Regulation of Trophoblast and Decidual Stromal Cells on the Function of Decidual NK cells through CollagenObjectives To prove the regulation of the trophoblast and decidual stromal cells from human first trimester pregnancy on the function of decidual NK cells through collagen.Methods Human trophoblast and DSCs were isolated and identified as our previous study. After co-cultured respectively with trophoblast, DSCs or trophoblast-DSCs unit for 48 hours, the decidual NK cells were collected and analyzed by FCM. Then, trophoblast cell line HTR-8 and DSCs were tranfected with P4H shRNA plasmid. The same analysis was performed to the dNK cells after co-cultured with P4H shRNA plasmid transfected trophoblast and DSCs.Results PCR and ELISA identification showed that we got the successfully transfected HTR-8 and DSCs. We found that trophoblast HTR-8 and DSCs could up regulate the expression of KIR2DL1 and down regulate the expression of NKp30, IFN-y and TNF-a through collagens. There was also a decrease in the IL-4 and IL-10 expression after P4H shRNA treatment in the co-culture system.Conclusions We found that trophoblast HTR-8 and DSCs could modulate the phenotype, Th1/Th2 cytokine and perforin expression of decidual NK cells through collagens, which contributes to maternal-fetal immune tolerance for the successful pregnancy.Part IV JAK-STAT Signaling Pathway is Involved in the Regulation of LAIR-1 on NK cellsObjectives To analyze the signaling pathway involved in the regulation of LAIR-1/collagens interaction on the function of NK cells.Methods NK cells were isolated and purified from normal early pregnancy woman’s peripheral blood and normal early pregnancy deciduas. After co-cultured with collagens, the culture medium from the supernant of trophoblast and DSCs was added into the NK culture system. The NK cells in the supernant were collected and Western-blot was used to detect the expression of LAIR-1, STAT1, p-STAT1, STAT4, p-STAT4. What’s more, FCM was used to analyze the transcript factor in the downstream of JAK-STAT signaling pathway. CoIP was applied to analyze the related molecules in the upstream of JAK-STAT signaling pathway. Finally, we constructed the LAIR-1 over-expressed NK-92 cell lines with pCDH-CMV-MCS-EFl-copGFP plasmid and do the identification and analysis with the Q-PCR, Western-blot and fluorescent microscopy methods.Results LAIR-1 can bind SHP-1. SHP-1 can bind JAK1 and JAK2. The interaction of collagens and LAIR-1 inhibited the activation of STAT1 and STAT4, and in turn down regulate the expression of T-bet and Helios. The LAIR-1 over-expressed NK-92 cell line was constructed successfully.Conclusions JAK-STAT signaling pathway is involved in the regulation of LAIR-1/collagens interaction on the function of NK cells. The LAIR-1 over-expressed NK-92 cell line will be great valuable for the next research work.In this study, we clarified the mechanisms of maternal-fetal immune tolerance by focusing on the interactions between the LAIR family members and collagens upon the prominent decidual NK cells. Our research deepened our understanding of maternal-fetal immune regulation in the maternal-fetal interface. Our research will be valuable in innovating the new scientific evidence and idea for the prevention and therapy of pregnancy wastage.