Effects Of SEB Exposure To Pregnant Rats On Treg Cells In The Thymus And Peripheral Blood Of Their Offspring Rats

Background:The annual rate of birth defects is about 5.6%in China.The causes of birth defects are complex.Genetic factors and environmental factors can lead to birth defects.Microbial infection during pregnancy is one of the main types of environmental factors.Staphylococcus aureus(S.aureus)is one of the most common pathogens of bacterial infectious diseases,and it is also an important factor leading to adversing pregnancy outcomes and fetal development abnormalities in pregnant women.Staphylococcal enterotoxin B(SEB)is one of the important pathogenic substances produced by S.aureus.It is not only a toxic pathogenic substance but also an important superantigen in the superantigen family.Our previous studies found that SEB administration in pregnant rats significantly affected the proportion of CD4/CD8 T cells in the central and peripheral regions of offspring rats.However,it is unknown about the effects of SEB administration in pregnant rats on the offspring rats of CD4~+CD25~+FoxP3~+Treg cells(hereinafter referred to as“Treg cells”)which are the main cells to regulate the balance of T cells.The purpose of this study was to investigate the effects of SEB on Treg cells in the thymus and peripheral blood of neonatal and adult offspring rats born to the mothers injected by SEB during pregnancy.Flow cytometry was employed to detect the number of Treg cells in thymus and peripheral blood of neonatal and adult offspring rats.The contents of IL-10 and TGF-?in peripheral blood were determined by ELISA.The expression levels of FoxP3 mRNA and protein were detected by real-time quantitative PCR and western blot.The results will give important insight on exploring the relationship between SEB and immunological diseases as well as improving population health and birth quality.Objective:To study the effects of SEB exposure to pregnant rats on Treg cells in the thymus and peripheral blood of their offspring.Methods:1.Establishment of the pregnant rats model and SEB injectionAdult male and female SD rats(about 50 females and 20 males)were employed for this study.Each female rat was mated with a male rat at18:00 and checked each morning for the presence of a vaginal plug.Day1 of gestation(GD)was defined as the day when a plug was initially observed in the vagina.When the pregnancy was confirmed,the females were isolated from the males and kept in separate cages.Time-gated pregnant rats were randomly divided into the following two groups at GD16:the control[(phosphate buffered saline,PBS)]group and the SEB group.In the SEB group,the pregnant rats were intravenously injected once with 0.3 ml 50?g/ml SEB in 0.2 M PBS.The pregnant rats in PBS group were intravenously injected once with the same volume of PBS.They were allowed to give birth naturally.Some neonatal offspring rats at day 1,3,5 after delivery were used to analyze the Treg cells in the thymus and peripheral blood,the others were reared to adulthood(about 3to 5 months)for the analysis of the Treg cells in the thymus and peripheral blood for the following experiments.2.Experimental groups2.1 In the part of newborn offspring rats,the studies were divided into experimental group and control group.The experimental group was that neonatal rats were delivered by pregnant rats given SEB injection on GD16,named as“neonatal SEB group”.The control group was that neonatal rats came from pregnant rats injected with the same volume of PBS,named as“neonatal PBS group”.2.2 For the part of adult offspring rats,the studies were divided into an experimental group and a control group.The experimental group was that adult offspring rats were born to the mothers injected by SEB during pregnancy,named as“adult SEB group”.The control group was that adult offspring rats were born to the mothers injected by PBS during pregnancy,named as“adult PBS group”.2.3 Studies for the adult offspring rats responded again to SEB were divided into four groups.Adult offspring rats born to the mothers injected by PBS during pregnancy were injected respectively with PBS or SEB during adulthood,which were correspondingly recorded as“PBS+PBS group”and“PBS+SEB group”.Adult offspring rats born to the mothers injected by SEB during pregnancy were respectively injected with PBS or SEB during adulthood,which were correspondingly recorded as“SEB+PBS group”“and“SEB+SEB group”.3.Study on the effects of SEB exposure to pregnant rats on Treg cells in the neonatal offspring rats3.1 Effects of SEB exposure to pregnant rats on the numbers of Treg cells in thymus and peripheral blood of neonatal offspring ratsPregnant rats were exposed to SEB during pregnancy.At day 1,3 and 5after the birth of newborn rats,their thymi and peripheral blood were acquired and isolated for mononuclear cells with lymphocyte separation solution.Then those cells were stained with fluorescent-labeled antibodies(CD3-FITC,CD4-APC,CD25-PE,FoxP3-percy5.5)and were determined by Flow cytometry for the proportions of Treg cells in thymus and peripheral blood.Lastly the absolute numbers of Treg cells were calculated.3.2 Effects of SEB exposure to pregnant rats on cytokines in peripheral blood of neonatal offspring ratsPregnant rats were injected with SEB during pregnancy.The anticoagulant blood was obtained from the newborn rats at the 5th day after birth,and was centrifuged to obtain plasma.Then the contents of IL-10 and TGF-?in the plasma were determined by ELISA.3.3 Effects of SEB exposure to pregnant rats on expression levels of FoxP3 protein and mRNA in neonatal offspring ratsMononuclear cells were obtained from thymi and peripheral blood of neonatal offspring rats.Their total RNA was extracted and reversely transcribed into cDNA,and the mRNA level of FoxP3 in the cells was detected by real-time PCR.The FoxP3 protein expression level was quantitatively detected by western blot after total protein was extracted from the samples.4.Effects of SEB exposure to pregnant rats on Treg cells in adult offspring rats4.1 Effects of SEB exposure to pregnant rats on the number of Treg cells in thymus and peripheral blood of adult offspring ratsAfter the pregnant rats were exposed to SEB during pregnancy,the neonatal rats were allowed to grow into adults(3-5 months).Their thymi and peripheral blood were acquired and isolated for mononuclear cells with lymphocyte separation solution.After those cells were stained with fluorescent-labeled antibodies(CD3-FITC,CD4-APC,CD25-PE,FoxP3-percy5.5),the proportions of Treg cells were measured by flow cytometry and the absolute numbers of Treg cells were calculated in thymus and peripheral blood of adult offspring rats.4.2 Effects of SEB exposure to pregnant rats on cytokines in peripheral blood of adult offspring ratsPregnant rats were injected with SEB during pregnancy.When the newborn rats grew into adults,their blood was extracted from the abdominal aorta and anticoagulated.Plasma was obtained from their blood after centrifugation,and the contents of IL-10 and TGF-?were determined by ELISA.4.3 Effects of SEB exposure to pregnant rats on expression levels of FoxP3 protein and mRNA in adult offspring ratsMononuclear cells were obtained from thymus and peripheral blood of adult offspring rats.Total RNA was extracted and reversely transcribed into cDNA.The mRNA level of FoxP3 in these cells was detected by real-time PCR.The protein expression level of FoxP3 was quantitatively detected by western blot after total protein was extracted from the samples.5.Effects of SEB re-administration on Treg cells of adult offspring rats Adult offspring rats born to pregnant rats injected by SEB or PBS during pregnancy were given SEB or PBS by tail vein injection.After 5 days of injection,the thymus and peripheral blood of adult offspring rats were obtained for the following study.5.1 Effects of SEB re-administration on the numbers of Treg cells in thymus and peripheral blood of adult offspring ratsLymphocyte assay was used to separate mononuclear cells in thymus and peripheral blood of adult offspring rats.These cells were used for intracellular and extracellular staining with fluorescent-labeled antibodies.The proportions of Treg cells were determined by flow cytometry and the absolute numbers of Treg cells were calculated in thymus and peripheral blood of adult offspring rats.5.2 Effects of SEB re-administration on cytokines in peripheral blood of adult offspring ratsPlasma was obtained after centrifugation of anticoagulant peripheral blood,and the contents of IL-10 and TGF-?in adult offspring rats were determined by ELISA.5.3 Effects of SEB re-administration on expression levels of FoxP3protein and mRNA in adult offspring ratsTotal RNA was extracted from mononuclear cells in thymus and peripheral blood of adult offspring rats,and their total RNA was reverse-transcribed into cDNA.The mRNA level of FoxP3 in these cells was detected by real-time PCR.The protein expression level of FoxP3was quantitatively detected by western blot after total protein was extracted from the samples.Results:1.The model of pregnant rats was established successfullyFemale rats were considered to be pregnant when a vaginal plug was found in the next morning.Pregnant rats was injected 15?g SEB via the tail vein on GD16.Then the pregnant rats were reared and allowed to delivery naturally.This indicated that the model of the pregnant rats was established successfully with the rate of 70-80%.2.Effects of SEB exposure to pregnant rats during pregnancy on Treg cells in neonatal offspring rats(1)SEB exposure to pregnant rats during pregnancy had no effect on the percentages of Treg cells in thymus and peripheral blood of neonatal offspring rats,but the absolute numbers of Treg cells were significantly increased in thymus and peripheral blood of neonatal offspring rats at day1,3,5 after delivery.(2)SEB exposure to pregnant rats during pregnancy increased significantly the levels of IL-10 and TGF-?in peripheral blood of neonatal offspring rats at day 5 after delivery.(3)SEB exposure to pregnant rats during pregnancy increased significantly the expression levels of FoxP3 mRNA and protein in thymus and peripheral blood of neonatal offspring rats at day 1,3,5 after delivery.3.Effects of SEB exposure to pregnant rats during pregnancy on Treg cells in adult offspring rats(1)The percentages of Treg cells in thymus and peripheral blood of adult offspring rats was not affected by the exposure of pregnant rats to SEB,but the absolute numbers of Treg cells were significantly increased.(2)SEB exposure to pregnant rats during pregnancy increased significantly the levels of IL-10 and TGF-?in peripheral blood of adult offspring rats.(3)SEB exposure to pregnant rats during pregnancy increased significantly the expression levels of FoxP3 mRNA and protein in thymus and peripheral blood of adult offspring rats.4.Effects of SEB re-administration on Treg cells of adult offspring rats born to pregnant rats injected with SEB during pregnancy(1)The proportion of Treg cells in thymus was significantly higher in PBS+SEB group than that in PBS+PBS group,while the proportion of Treg cells in thymus was significantly lower in SEB+SEB group than that in PBS+SEB group.The absolute numbers of Treg cells in thymus increased significantly between groups of SEB+PBS and PBS+PBS,SEB+SEB and PBS+SEB,while the absolute numbers of Treg cells decreased significantly between groups of PBS+SEB and PBS+PBS,SEB+SEB and SEB+PBS.The absolute numbers of Treg cells in peripheral blood increased significantly in PBS+SEB group compared with PBS+PBS group,while the absolute numbers of Treg cells in peripheral blood decreased significantly in SEB+SEB group compared with PBS+SEB group.Among other groups,the changes in the proportion and absolute number of Treg cells in peripheral blood were consistent with the changes in the thymus.(2)Compared with PBS+PBS,the levels of IL-10 and TGF-?in peripheral blood increased significantly in SEB+PBS and PBS+SEB group,while the levels of IL-10 and TGF-?in peripheral blood was significantly lower in SEB+SEB group than those of PBS+SEB and SEB+PBS groups.(3)Compared with PBS+PBS,the expression levels of FoxP3 mRNA and protein in thymus and peripheral blood increased significantly in SEB+PBS and PBS+SEB group,while the expression levels of FoxP3mRNA and protein in thymus and peripheral blood were significantly lower in SEB+SEB group than those of PBS+SEB and SEB+PBS groups.Conclusion:1.SEB exposure to pregnant rats during pregnancy could lead to the effects on Treg cells number,FoxP3 and cytokines in the thymus and peripheral blood of neonatal offspring rats.2.SEB exposure to pregnant rats during pregnancy could cause the significant changes on Treg cells number,FoxP3 and cytokines in the thymus and peripheral blood of adult offspring rats.Furthermore,these changes in adult offspring rats were consistent with those in neonatal offspring rats.Those results suggest that the effects of SEB exposure to pregnant rats on Treg cell in offspring rats could be retained from the neonatal stage to the adult stage and could cause the imprinting effect.3.SEB exposure to pregnant rats during pregnancy could change the response ability of central and peripheral Treg cells to SEB re-administration in adult offspring rats.