Effects Of Prenatal SEB Administration To Pregnant Rats On Hedgehog Signaling Pathway In Thymic And Splenic T Cells Of The Offspring And Its Relationship With Splenic T Cells

Background:According to the statistics of the"China birth defects prevention and control report"released by the National Health Commission,the birth defect rate of newborns is about 5.6%in China.The etiology to cause birth defects is complex and multiple,mainly including genetic factors,environmental factors,and their combination.Microbial infection during pregnancy is a common environmental factor.Staphylococcus aureus(S.aureus)can colonize in the skin,mucous membranes,reproductive tract and other parts of healthy individuals.S.aureus is one of the most common pathogens that cause clinical bacterial infectious diseases such as pyogenic inflammation,sepsis,food poisoning and other diseases.Current studies have found that S.aureus is also an important factor to cause adverse pregnancy outcomes and abnormal fetal development.Staphylococcal enterotoxin B(SEB)is one of the important pathogenic substances produced by S.aureus,and also is an important superantigen.In contrast to conventional antigens,the SEB can lead to the non-specific activation of lots of T cells,which does not require the antigen processing and presentation by antigen presenting cells.A large number of the activated T cells by SEB are eliminated under certain condition to cause the abnormalities in the number and function of T cells which results in the immunosuppression of host after microbial infection.Hedgehog(Hh)signaling pathway which plays an important role in the development process from embryo to adult is closely related to embryonic development,cell proliferation and differentiation,tissue formation and repair,as well as immune regulation.Hh signaling pathway not only participates in the formation of T cell in the body and the regulation of its different developmental stages,but also regulates the proliferation,activation and function of peripheral T cells,which reveals that Hh signaling pathway plays a more important role in the response process of T cells.The preliminary results by my supervisor found that the prenatal exposure of pregnant rats to SEB could influence on the number and function of T cells in newborn and adult offspring,but it has not been reported so far whether the cellular immunity affected by the prenatal exposure to SEB is caused by the Hh signaling pathway.Therefore,we propose the hypothesis that the effect of exposure of pregnant rats to SEB on the Hedgehog signaling pathway in thymic and splenic T cells of the offspring and its relationship with splenic T cells.In current study,the protein and m RNA levels of the ligands,key molecules and target genes of Hh signaling pathway in thymic and splenic T cells of the offspring were investigated by the methods of western blot,real-time PCR,immunomagnetic separation,immunofluorescence assay and cell culture.Meantime,it is to explore the relationship between T cell differentiation and the inhibited Hh signaling pathway.We hope to provide an experimental and theoretical basis for eugenics and fetal-borne diseases via elucidating these problems.Objective:To study the effects of prenatal SEB administration to pregnant rats on Hedgehog signaling pathway in thymic and splenic T cells of the offspring and its relationship with splenic T cells.Methods:(1)SEB injection and groups.Pregnant rats at GD16 were intravenously injected 0.3 ml50μg/ml SEB,which was recorded as SEB group.Meantime,other pregnant rats were given PBS as control group.Then,these pregnant rats continued to be reared and were allowed to natural delivery.After the natural delivery,a part of the newborn offspring were chosen for the following studies at 14 days in life,and the other part were reared to the adulthood(10 weeks after birth)for the corresponding studies.The thymus and spleen of newborn and adult offspring were aseptically harvested for the experimental study as follow.(2)It was extracted the total RNA and total protein from the thymus or spleen of neonatal and adult offspring in PBS and SEB groups.Then the expression level of ligands in Hedgehog signaling pathway was detected by the methods of real-time PCR and western blot.(3)The single cell suspension of thymus tissue was prepared with 200-mm mesh screen by pressing thymic tissue from the neonatal and adult offspring in PBS and SEB groups.Then mononuclear cells were isolated through lymphocyte separation medium and their total RNA and total protein were extracted.The methods of real-time PCR and western blot were employed to determine the expression levels of ligands,receptors,transcription factors and target proteins in Hedgehog signaling pathway.(4)The single cell suspension of spleen tissue was prepared with 200-mm mesh screen by pressing spleen tissue of neonatal and adult offspring in PBS and SEB groups.The mononuclear cells were isolated with lymphocyte separation medium.The CD4~+T cells and the CD8~+T cells from mononuclear cells were sorted by MACS magnetic bead analyzer,and their total RNA and total protein were extracted.Then the expression levels of ligands,receptors,transcription factors and target proteins in Hedgehog signaling pathway were detected by the methods of real-time PCR and western blot.(5)Immunofluorescence staining was used to monitor the expression and localization of Gli1 in thymic lymphocytes and spleen CD4~+T cells and CD8~+T cells in PBS and SEB groups.(6)Twenty-four hours after the splenic CD4~+T cells in adult offspring were co-cultured with inhibitor Gant61,the expression levels of effective and target protein in both Hedgehog signaling pathway and Wnt/β-catenin signaling pathway were detected by western blot among PBS,SEB,inhibitor+PBS and inhibitor+SEB groups.(7)After the lymphocytes from adult offspring spleen were acquired in PBS and SEB groups,and were co-cultured for 24h with inhibitor Gant61,flow cytometry was used to detect the percentages of both CD4~+T and CD8~+T cells.Results:(1)Compared with PBS group,the expression levels of ligands Shh and Dhh in Hh signaling pathway significantly increased in thymus tissue of the neonatal offspring in SEB group.(2)Exposure of pregnant rats to SEB significantly decreased the expression levels of membrane receptors(Ptch1,Smo),transcription factor Gli1,as well as target genes(Cyclin D1,C-myc and N-myc)in Hh signaling pathway in thymic T lymphocytes of the neonatal offspring.(3)Exposure of pregnant rats to SEB notedly increased the expression levels of ligands Shh and Dhh in Hh signaling pathway in thymus tissue of the adult offspring.(4)The expression levels of membrane receptors(Ptch1,Smo),transcription factor Gli1and target genes(Bcl2,Cyclin D1,C-myc and N-myc)in Hh signaling pathway were significantly lower in SEB group than those in PBS group in thymic T lymphocytes of the adult offspring.(5)Exposure of pregnant rats to SEB clearly increased the expression levels of ligands Shh and Dhh in Hh signaling pathway in spleen tissue of the neonatal offspring.(6)Compared with PBS group,exposure of pregnant rats to SEB remarkably increased the expression of membrane receptors(Ptch1,Smo and Boc),transcription factor Gli1,target genes(Bcl2,Cyclin D1 and N-myc)in Hh signaling pathway in splenic CD4~+T cells of the neonatal offspring.(7)Exposure of pregnant rats to SEB significantly increased the expression levels of ligands Shh and Dhh in Hh signaling pathway in spleen tissue of the adult offspring.(8)In CD4~+T cells of adult offspring,the expression levels of Hedgehog signaling pathway membrane receptors(Ptch1 and Smo),transcription factor Gli1,and target genes(Cyclin D1,N-myc and C-myc)were significantly higher in SEB group than those in PBS group.(9)Exposure of pregnant rats to SEB did not affect the expression levels of downstream receptors(Ptch1,Smo,Boc and Cdon),transcription factor(Gli1,Gli2 and Gli3),and target genes(Bcl2,N-myc,Cyclin D1 and Vegfa)in Hh signaling pathway in splenic CD8~+T cells of the adult offspring,but reduced the expression level of C-myc protein.(10)After the splenic CD4~+T cells in adult offspring were co-cultured with inhibitor Gant61,the Gant61 significantly reduced the expression level of transcription factor Gli1 and significantly increased target proteins of Bcl2 and N-myc in Hh signaling pathway in PBS or SEB group.The inhibitor Gant61 also led to the decreased expression of target proteins Cyclin D1 and C-myc in Wnt signaling pathway.The inhibited impact by Gant61 on the expression levels of Gli1,Cyclin D1 and C-myc proteins in SEB group was significantly higher than those in PBS group.(11)After the splenic T lymphocytes in adult offspring were co-cultured with inhibitor Gant61,the Gant61 notedly reduced the percentages of CD4~+T cells and CD8~+T cells and increased the percentage of CD4~-CD8~-T cells in PBS or SEB group.However,the inhibitor did not change the reduced rates of CD4~+or CD8~+T cells between PBS group and SEB group.Conclusion:1.Exposure of pregnant rats to SEB can inhibit Hh signaling pathway in thymic T cells in the newborn offspring.This effect can maintain to adult offspring,which is termed imprinting effect.2.Exposure of pregnant rats to SEB activates Hedgehog signaling pathway in splenic CD4~+T cells in the newborn offspring and this activation forms an imprinting effect that keeps to adult offspring.But exposure of pregnant rats to SEB has not affect Hedgehog signaling pathway in the splenic CD8~+T cells,which suggest that exposure of pregnant rats to SEB has cellular selectivity to activate Hedgehog signaling pathway in splenic T cells of the offspring.3.Exposure of pregnant rats to SEB has different influences on the Hedgehog signaling pathway between the central immune organ thymus and the peripheral immune organ spleen in the offspring.4.Exposure of pregnant rats to SEB strengthens the responsibility of Hedgehog signaling pathway to inhibitor in CD4~+T cells of the adult offspring spleen,but it does not change the inhibition effect by the inhibitor on CD4~+T and CD8~+T subsets in adult offspring spleen,suggesting that the effects of exposure of pregnant rats to SEB on splenic T cells are not associated with the activated Hedgehog signaling pathway.