Expression Of Human Thymic Stromal Lymphopoietin At Materno-fetal Interface And Its Regulation In Materno-fetal Immunotolerance

The embryo expresses antigens foreign to the mother,and therefore has been viewed as allograft.Induction of maternal tolerance to persistence of the alloantigen is a main purpose for maintenance of pregnancy.It has been proven that the materno-fetal interface exhibits a Th2 bias,including IL-4,IL-5 and IL-10 secretion during pregnancy.Failure to establish such an immune milieu or Th1 bias such as interferon(IFN)-γand tumor necrosis factor(TNF)-αis associated with fetal miscarriage.Regulatroy T cells(Treg) play an important role in preventing from autoimmune reaction and rejection of the transplantation. It has been found that Treg expands during pregnancy,but the mechanism underlining the expansion has to be elucidated.Thymic stromal lymphopoietin(TSLP) is an IL-7-like cytokine,originally cloned from a murine thymic stromal cell line Z210R.1 that supports the development B cells.The murine TSLP receptor(TSLP-R) consists of the IL-7 receptorα(IL-7Rα) chain and a commonγreceptor(γ_c)-like chain,TSLPR-γ.Human TSLP(hTSLP) and hTSLPRγwere cloned in 2001 by computational analyses of human genomic data.The hTSLP is produced generally by epithelial cells such as skin keratinocytes,tonsil crypt epithelium,bronchial epithelial cells,and mediates such allergic inflammation as asthma and atopic dermatitis. The TSLP-R is mainly expressed on dendritic cells(DCs),and TSLP activates DCs via TSLP-TSLPR interaction,and induces a Th2 type inflammatory response through OX40 ligand.Not completely consistent with the situation of allergic diseases,in human intestine, Mo-DCs from PBMC or gut DCs activated by mucosal epithelial cells produce a classical Th2 response with high levels of IL-4 and IL-10,but low levels of IFN-γand IL-12. However,whether there is a TSLP-TSLP-R signaling pathway,and how it acts at the materno-fetal interface remains unknown.In the present study,we propose to investigate the expression and regulation of TSLP and its receptor at human materno-fetal interface.PartⅠThe expression of hTSLP at human materno-fetal interfaceObjective To investigate whether TSLP is expressed at human materno-fetal interface and how its expression is regulated.Methods RT-PCR,real-time RT-PCR,immunochemistry and traditional western blot was used,respectively,to study the relative expression level of TSLP in villi and decidua from unexplained miscarriage with normal early pregnancy as control.We also investigated the effect of pregnancy-associated hormones and Th2/Th1 cytokines on the expression of TSLP in human trophoblast.Results All the deciduae and villi from normal pregnancy,as well as endometrial tissues of non-pregnany,expressed TSLP mRNA.Immunochemistry analysis showed that the villous and decidual tissues from normal and abortive pregnancies were all positively stained with anti-TSLP antibody,but negative in endometrium of fertile women without pregnancy.The trophoblasts and DECs were positive for TSLP,while the decidualized DSCs were not.The villi and deciduae from normal early pregnancy had a significantly higher TSLP transcription level than that of the unexplained miscarriage.TSLP in supematant was about 19.30 pg/ml when the normal trophoblasts were seeded 1×10~6 cells/ml/well and cultured for 72 h.Combination of progesterone and 17β-estrodial withβ-hCG increased TSLP mRNA expression to 12-fold in trophoblasts,and the TSLP secretory level to 28.23pg/ml by human trophoblasts.The combination of TNF-αand IL-4 increased the TSLP mRNA nearly 2 fold, while the TGF-β1 showed little effect on the expression of TSLP by trophoblasts.Conclusions hTSLP is expressed at human matemo-fetal interface,and is mainly secreted by trophoblasts.Normal pregnancy had a much higher TSLP expression level than the unexplained miscarriage.Combination of progesterone and 17β-estrodial withβ-hCG enhanced TSLP mRNA expression in trophoblast.PartⅡhTSLP improves proliferation and invasion of trophoblast via TSLP/TSLP-R axis in an autocrine mannerObjective To study whether the TSLP secreted by trophoblasts could affect viability and invasion themselves.Methods Immunocytochemistry was done on the purified trophoblast to detect the TSLP-R.Trophoblast of the first-trimester were stimulated with rhTSLP(0,5,50,100,200, 400ng/ml) for 24-48 h,and the proliferation was detected by[~3H]thymicidine incorporation and WST-8 production formazan optical density(Cell Counting Kit-8),respectively.The trophoblast invasion was observed with graded concentrations of rhTSLP treatments. Antibody neutralizing TSLP was used to interfere with TSLP action in these processes.Results Human trophoblast of the early pregnancy expressed TSLP and TSLPR simultaneously.The rhTSLP(100-400 ng/ml) could significantly increase the viability and invasiveness of trophoblast in a dose-dependent manner.It was also found that antibody neutralizing TSLP could significantly abolish the enhanced viability and invasiveness of human trophoblast.Conclusions TSLP improves the the proliferation and invasiveness of trophoblasts in an autocrine manner.PartⅢhTSLP instructed dDCs to induce a Th2 bias at materno-fetal a interfaceObjectives To investigate the regulation of TSLP at materno-fetal interface on dDCs and then the TSLP-dDCs on the dCD4~+T cells.Methods dDCs were isolated from the trypsin and DNAse-digested decidua by MACS with the CDlc marker.The dDCs were stimulated with rhTSLP or cocultured with human early pregnancy trophoblast,and the cytokines in the supematants were detected by ELISA.The TSLP-activated dDCs(TSLP-dDCs) were then co-cultured with allogeneic or autologous dCD4~+T cells,and the cytokines in the supematants was determined by ELISA. The transcriptional factors T-bet,GATA-3 were quantitatively measured by real-time PCRResults TSLP stimulated dDCs to secret high levels of IL-10 and chemokine CCL17, but low level of TNF-α.When the dDCs were cocultured with trophoblasts in the absence of antibody neutralizing TSLP,the levels of IL-10,CCL17 and TNF-αin the culture supematant were similar to the rhTSLP stimulation in vitro.But the neutralizing antibody reversed the dDCs to secrete these cytokines,which indicates that TSLP plays a key role in the Th2 response at the matemo-fetal interface via the dDCs.The TSLP-dDCs could induce the dCD4~+T cells to secret high levels of IL-4 and IL-10,but low levels of TNF-αand IFN-γ.The dCD4~+ T cells primed by TSLP-dDCs expressed higher level of GATA-3.Conclusions The rhTSLP can activate the dDCs to secret high levels of IL-10 and CCL17 and low level of TNF-α,and the TSLP-dDCs then induced Th2 bias and higher GATA-3 transcription at the materno-fetal interface.PartⅣTSLP-dDCs induced dCD4~+T cells to differentiate into CD4~+Foxp3~+ Treg at materno-fetal interfaceObjectives The mehcanism of the expanded Treg in the early pregnancy remians unclear,we proposed to investigate whether hTSLP secreted by trophoblast was involved in the differentiation of the dCD4~+T cells into CD4+Foxp3+Treg at materno-fetal interface.Methods TSLP-dDCs were co-culture with dCD4~+T cells for 7 days,and dCD4~+T cells were analyzed for Foxp3 expression by FCM.The suppressive funciton of the induced dCD4~+Foxp3~+Treg were analyzed by[~3H]thymicidine incorporation.Results TSLP induced a vigorous expansion in the total number of decidual monocytes cells in absense of uNK cells,whereas the unstimulated dDCs or dDCs exposed to IL-7 or LPS did not.After 7 days of co-culture of the dCD4~+CD25~- T cells with TSLP-dDCs,the CD4~+Foxp3~+T cells were apparently expanded.Both the freshly isolated CD4~+CD25~+ Treg and the induced CD4~+CD25~+ T cells showed little response upon anti-CD3 and anti-CD28 stimulation,and could inhibit the dCD4~+CD25~- T cells to proliferate upon the stimulation.Antibody neutralizing TGF-β1 partly inhibited the Treg differentiation induced by the TSLP-dDCs.Conclusions The TSLP-dDCs induce dCD4~+T cells to differentiate into CD4~+Foxp3~+ Treg cells at the matemo-fetal interface.TGF-βis involved in this process.The induced CD4~+Foxp3~+ Treg has the similar suppressive function on the dCD4~+CD25~- T cells with the natural dCD4~+Foxp3~+ Treg.In summary,the trophoblasts express functional TSLP,which increases the proliferation and invasiveness of human trophoblast cells through an autocrine fashion. Quantitative RT-PCR and westem blot analysis indicate that the villi and trophoblasts from normal pregnancy exhibit significantly higher levels of TSLP than that of the unexplained miscarriage,dDCs cocultured with trophoblasts or their supematant result in a high level of IL-10 and CCL 17 secretion,and a lower level of TNF-α.The TSLP-dDCs strongly polarize the dCD4~+T cells toward a classical Th2 bias,which secrets more IL-4 and IL-10,and less IFN-γand TNF-α.TSLP-dDCs also induce the autologous dCD4~+T cells to differentiate into CD4~+Foxp3~+ Treg,which implies that human trophoblast instruct the dDCs to induce Th2 and Treg differentiation at the materno-fetal interface,leading to the normal pregnancy smoothly.