Effect Of IDO Expression At The Early Pregnancy Fetal-Maternal Interface On Decidual NK Cells And Dentritic Cells

In human pregnancy,the implanted embryo constitutes a hemiallograft but remains spared from attack by the maternal immune system,suggesting a tolerance of its fetus at the site of contact between mother and child.Serving as an immunologically privileged tissue,the decidua and its components,especially decidual leucocytes,play essential functions in pregnancy maintenance.The decidual leucocyte population has been a centre of interest for the understandingof the mechanism that might control maternal immune responses in successful pregnancy. During the first trimester of pregnancy,the human decidua is rich in leucocytes which make up 10-15%of all decidual cells.This leucocyte population is composed of 70% decidual natural killer(dNK)cells,10%T cells and 20%major histocompatibility complex(MHC)classâ…¡-positive antigen-presenting cells(APCs)which are thought to be mainly macrophages and dendritic cells(DCs)Indoleamine 2,3-dioxygenase(IDO)has been found in placenta,decidual dendritic cells,monocytes and glandular epithelium.IDO is a heme-containing rate-limiting enzyme,which catalyzes the conversion of tryptophan to kynurenine,the main tryptophan metabolite.Tryptophan is the least available essential amino acid and is required by all forms of life for protein synthesis and other important metabolic functions.IDO-generated low tryptophan environment may inhibit T cell and NK cells proliferation and induce tolerogenic DCs,and play a key role in pregnancy maintance.In 1998,Munn et al.showed that murine placenta is protected from immune rejection by maternal T cells by means of localized IDO dependent depletion of tryptophan.Following work from the same group suggested that rejection of the allogeneic fetus is accompanied by a unique form of inflammation that is characterised by T cell dependent and antibody independent activation of complement. IDO synthesis is necessary to maintain effective immunological protection during gestation.While the mechanisms of IDO-dependent inhibition of T-cell function have been elucidated,less is known on the possible role of IDO in regulating natural killer (NK)-cell activity and DCs functions.Decidual NK cells has unique phenotypical and functional characteristcs,and the phenocyte of 90%Decidual NK cells is CD56^birghtCD16^- and less than 10%of dNK is CD56^dimCD16⁺.Human CD56^birghtNK cells accumulate in the maternal decidua during first trimester of pregnancy and are found in direct contact with fetal trophoblasts.It is currently believed that in the early stages of gestation,dNK cells may play an important role in the control of immune tolerance at the fetal-maternal interface and trophoblastic growth,differentiation,and invasion.NK-cell function is regulated by a balance between activating and inhibitory signals.Decidual NK cells can express natural cytotoxicity receptors(NCRs),such as NKp46,NKp44,NKp30 and NKG2D.Transcriptional geneexpression profiling has shown that dNK cells express both perforin and granzymes at a similar or even higher level than CD56^dim CD 16⁺ pNK cells.These observations suggested that dNK cells might have cytotoxic potential.During normal pregnancy,dNK cells remain cytotoxicity at a low level. However,abnormal expression and function of activating recepors may lead to pregnancy loss.It has recently been shown that the tryptophan catabolite L-kynurenine inhibits the surface expression of NKp46-and NKG2D-activating receptors and regulates NK-cell function.Also IDO-generated L-kynurenine,in addition to T-cell proliferation,is also able to inhibit the IL-2-induced proliferation of NK cells.The effect of IDO expressed at the fetal-maternal interface is still largely unknown.We formulated the hypothesis that IDO expression at the fetal-maternal interface may inhibit the surface expression of NKp46-and NKG2D-activating receptors and regulate NK-cell function.To study the mechanism that control the cytotoxicity of dNK cells may help to understand the mechanism of immune tolerance at the fetal-maternal ineraction.Interestingly,the human decidua was described recently to harbour DCs,which has pointed to a critical role of DCs at the fetal-maternal interface.DCs can acquire unique features or phenotypes in different tissue microenvironments,and different DC subsets may play a prominent role in dictating the quantity and quality of immune responses and decide whether immunity or tolerance develops.Accumulating evidence suggests that DCs in situ can induce antigenspecific unresponsiveness or tolerance in central lymphoid organs and in the periphery.DCs can induce maternal tolerance by up-regulating IDO expression.It was reported that IDO expression in decidual monocytes and DCs by CTLA-4/Fc or IFN-γtreatment were increased in therapeutic abortion decidua but were decreased in spontaneous abortion,suggesting the key role of IDO expression on DCs.In the past,the investigation of DC subsets in the human decidua has been hampered by the lack of specific markers identifying DCs directly and by the scarcity of DCs.Several groups have reported the presence of certain DC subsets at the fetal-maternal interface;however,the precise distributional and phenotypic characteristics of DC subsets in the human decidua are still poorly understood.The study to the effect of IDO expression on the function of dNK Cells and the distribution and phenotypes of decidual DCs will provide new clue for understanding of physical and pathological reproduction and new thread for diagnosis and therapy of reproductive immune disease.PARTâ… RELATIONSHIP OF RECURRENT SPONTANEOUS ABORTION AND THE INDOLEAMINE 2,3-DIOXYGENASE EXPRESSION AT THE FETAL -MATERNAL INTERFACE AND HTR-8/SVNEO CELLSObjective:To study the relationship of recurrent spontaneous abortion(RSA) and the expression of IDO in villi,decidua and HTR-8/SVneo cells in vitro.Methods:(1)Immunohistochemistry was applied to analyze the expression of IDO protein in villus and decidua from normal pregnancy and RSA.Quantitative RT-PCR was applied to analyze the mRNA transcription of IDO.(2)Human trophoblast villous explant was cultured,and highperformance liquid chromatography (HPLC)was applied to determinate whether there was kynurenine in culture medium of trophoblast villous explant and the effect of IFN-γon the mRNA expression and activity of IDO.(3)Immunohistochemistry was applied to analyze the expression of IDO protein in HTR-8/SVneo cells.Quantitative RT-PCR was applied to analyze the mRNA transcription of IDO.HPLC was applied to determinate whether there was kynurenine in culture medium of cells and the effect of IFN-γon the mRNA expression and activity of IDO.Results:(1)IDO protein was expressed in villous syncytiotrophoblast,EVT cell columns and decidual gland.In minor patients,IDO protein was expressed in cytotrophoblast and EVT cell column,but syncytiotrophoblast.IDO mRNA was detected in villus and decidua.(2)Human trophoblast villous explant ultured for 48 h was used to determine IDO activity by HPLC.The level of IDO activity treated with IFN-γwas significantly higher than that of control(P＜0.05).(3)IDO protein and mRNA were found to be expressed in HTR-8/SVneo cells,and there was kynurenine in the cell culture medium.(4)The expression of IDO protein and mRNA in villus and decidua from RSA were lower than that from normal pregnancy.Conclusion:Appropriate expression of IDO at the fetal-maternal interface is necessary for maintenance of normal pregnancy,and an active IDO protein was expresseed in HTR-8/SVneo cells.PARTâ…¡THE EFFECT OF INDOLEAMINE 2,3-DIOXYGENASE EXPRESSED IN HTR-8/SVNEO CELLS ON THE SURFACE EXPRESSION OF NATURAL CYTOTOXICITY RECEPTORS AND THE FUNCTION OF DECIDUAL NK CELLSObjective:To study the effect of IDO expressed in HTR-g/SVneo cells on the surface expression of NKG2D and NKp46 and the cytotoxicity of dNK cells and dNK cells.Methods:(1)Decidual mononuclear cells were isolated from 5-9 weeks' decidua obtained from clinically normal pregnancies in vitro.CD56⁺ dNK cells were purified by use of human CD56 MicroBeads.(2)Purity and phenotype of CD56⁺ NK cells were then analyzed by flow cytometry(FCM).(3)CD56⁺dNK cells were cultured for 48 hours with complete RPMI 1640 medium,IDO conditioned medium(CM)which were obtained from HTR-8/SVneo cells,and IDO + 1-MT CM which were obtained from HTR-8/SVneo cells.Then qRT-PCR and FCM were applied to analyze the mRNA transcription and protein expression of NKG2D and NKp46 on dNK cells and pNK cells.(4)The cytotoxicity of dNK cells and pNK cells obtained were assessed by LDH assays.(5)The culture supernatants were then collected and analyzed for the presence of TNF-αby using ELISA.Results:(1)CD56⁺ dNK cells were purified by use of human CD56 MicroBeads. (2)The purity of dNK cells was all over 95%.The phenotype of dNK cells was CD56^bright.(3)The expression of IDO mRNA and protein on CD56⁺ pNK cells cultured with IDO CM were significantly lower than the other two groups(P＜0.05). (4)The pNK cells cultured with IDO showed a redudced ability to kill K562 cells than the other two groups(P＜0.05).(5)The level of TNF-αin IDO group was significantly lower than that in the other two groups(P＜0.05).Conclusion:IDO expressed in HTR-8/SVneo cells was found to prevent the cytokine-mediated up-regulation of the expression and function of NKG2D and NKp46 responsible for the induction of NK-cell-mediated killing.PARTâ…¢BDCA-1(+),BDCA-2(+)AND BDCA-3(+) DENDRITIC CELLS IN EARLY HUMAN PREGNANCY DECIDUAObjective:The aim of this study was to identify and characterize DCs in the cell isolates obtained from normal human first-trimester decidua with the recently developed BDCA markers.Whether the number of these DC subsets changes with gestational age was also investigated. Methods:(1)A non-enzymatic method was used to isolate decidual mononuclear cells from 6-9 weeks' decidua(n = 44)obtained from clinically normal pregnancies. Flow cytometry was used to analyse the cell preparations in terms of staining for BDCA-1,CD14,CD19,BDCA-3,CD14,BDCA-2 and CD123.(2)To characterize further the phenotypes of fresh decidual MDC1 and MDC2,flow cytometric analysis was performed with a panel of MoAbs including HLA-DR,CD80,CD86 and Immunoglobulin-like transcript 3(ILT3).(3)Immunohistochemical staining was used to analyze the distribution of BDCA-1⁺ and BDCA-3⁺ cells in the decidua(n = 12).Results:(1)Using flow cytometry,we identified three DC subsets in normal human first-trimester decidua:BDCA-1⁺ CD19^- CD14^- myeloid DC type 1(MDC1), BDCA-3⁺ CD14^- myeloid DC type 2(MDC2)and BDCA-2⁺ CD123⁺ plasmacytoid DC(PDC).The percentage of MDC1 to mononuclear cells in the decidua was similar to that in the peripheral blood controls.The percentage of MDC2 in the decidua was significantly higher than that in the peripheral blood controls,whereas the percentage of PDC was significantly lower.(2)Both MDC1 and MDC2 subsets expressed HLA-DR,CD86 and CD80 at low levels,suggesting a characteristic of immature myeloid DCs.ILT3,suggested to be involved in immune tolerance induction,was also expressed on decidual MDC1 and MDC2 subsets.(3)In addition,as gestational age increased from 6 to 9 weeks,the numbers of MDC1 decreased but MDC2 increased significantly.(3)BDCA-1⁺ cells are present in the decidual stroma,BDCA-3⁺ cells are present in the decidual stroma near maternal vesses and glands.Conclusions:we have demonstrated the presence of the three previously unidentified BDCA+ DC subsets in normal human first-trimester decidua: BDCA-I⁺CD19^-CD14^-MDC1,BDCA-3⁺ CD14^- MDC2 and BDCA-2⁺ CD123⁺ PDC. The distributional and phenotypic characteristics of these decidual DC subsets may be relevant to the immune tolerance necessary for the maintenance of pregnancy. Therefore,this study provides a basis for further research of the roles of DCs in immunoregulation of human pregnancy at the fetal-maternal interface.