Imprinting Effect Of Maternal SEB Administration During Pregnancy On T Cells In The Offspring Rats And Its Mechanism

Objectrve：Staphylococcal enterotoxin B (SEB) is not only a toxin, but also asuperantigen. As an enterotoxin, a lower amount of SEB can cause the body seriousdiseases; while as a superantigen, it can stimulate the body’s immune system to result inthe activation and proliferation of T cells. SEB after entering the body can cause theactivation and proliferation of Vβ8+T cells in the earlier period, then it can induceapoptosis of excessively proliferated cells and cause clonal deletion, which results in theappearance of the central and peripheral tolerance. Our previous studies found thatmaternal SEB administration during pregnancy could cause the changed percentages ofT cell subpopulations in neonatal offspring rats, suggested that maternal SEBadministration during pregnancy could affect, in the neonatal period, the cellularimmunity of offspring rats. But it is not known whether these changes can be retainedfrom the neonatal period to adulthood and then form the imprinting effect. Therefore,based on the previous studies, we investigated the influences of maternal SEBadministration during pregnancy on the cellular immunity of adult offspring rats. Theseindexes were determined as follow: the subpopulations of CD4/CD8T cells in thethymus, spleen and blood, as well as their responses to secondary SEB stimulation, theproliferation response of in vitro cultured splenocytes to SEB, the levels of both IFN-γand IL-4in peripheral blood and the methylation levels of both IFN-γ and IL-4inspleens. These results will provide the experimental basis for the effects of maternalSEB administration during pregnancy on the cellular immunity of offspring rats, andgive insight into the fetal origin of adult disease.Methods:1. The time-gated pregnant SD rats were employed for the present study. At gestationalday16, some pregnant rats were intravenously injected with15μg SEB in0.3mlPBS (phosphate buffer) as experimental group (SEB group)， others wereintravenously injected with the same volume PBS as control group (PBS group).Then, the pregnant rats continued to be raised until natural birth, and neonataloffspring rats were grown up to adulthood, namely adult offspring rats. 2. The thymus, spleen and peripheral blood of adult offspring rats were acquired andprepared into their cell suspensions. The percentages of CD4/CD8T cells weredetected with flow cytometry after they were stained by fluorescent antibodies(CD3-FITC, CD8-PE, CD4-APC).3. Both thymus and peripheral blood of adult offspring rats were harvested and preparedinto their cell suspensions. The percentages of Vβ8.2T cells were determined withflow cytometry after they were stained by fluorescent antibodies (Vβ8.2-FITC、CD8-PE、CD3-APC).4. The adult offspring rats in both SEB and PBS groups were intravenously injectedwith15μg SEB in0.3ml PBS, at the same time, the adult offspring rats in same litterwere administrated with the same volume PBS as control. After5days, the thymus,spleen and peripheral blood were acquired and prepared into their cell suspensions.The percentages of CD4/CD8T cells were detected with flow cytometry after theywere stained by fluorescent antibodies (CD3-FITC, CD8-PE, CD4-APC).5. The adult offspring rats in both SEB and PBS groups were intravenously injectedwith15μg SEB in0.3ml PBS, at the same time, the adult offspring rats in same litterwere administrated with the same volume PBS as control. After5days, both thymusand peripheral blood were harvested and prepared into their cell suspensions. Thepercentages of Vβ8.2T cells were determined with flow cytometry after they werestained by fluorescent antibodies (Vβ8.2-FITC、CD8-PE、CD3-APC).6. In sterile conditions, the splenocytes of adult offspring rats were isolated andcontinuously co-cultured in vitro for3days with ConA and SEB, respectively. Then,both the proliferation index and the subpopulations of T cells were determined at eachday.7. The plasma of adult offspring rats was acquired and detected for the levels of bothIFN-γ and IL-4with ELISA.8. The spleens of adult offspring rats were harvested and detected for the methylationlevels of both IFN-γ and IL-4gene through the MeDIP-qPCR method.Results:1. Effect of maternal SEB administration during pregnancy on cellular immunity ofadult offspring rats(1) In the thymus, spleen and peripheral blood of both female and male adult offspringrats, maternal SEB administration during pregnancy significantly increased the percentage of CD4+CD8cells and reduced the percentage of CD4CD8+cells.(2) In both the thymus and peripheral blood of both female and male adult offspring rats,maternal SEB administration during pregnancy significantly decreased thepercentage of Vβ8.2T cells.2. Effect of secondary SEB administration on CD4/CD8T cells subpopulation in adultoffspring ratsContrast to the PBS control in same litter, secondary SEB administrationsignificantly reduced the percentage of CD4+CD8T cells, and increased the percentageof CD4CD8+T cells in the thymus. The changes of percentages of both CD4+CD8Tand CD4CD8+T cells in both spleen and peripheral blood were similar to those in thethymus.3. Effect of secondary SEB administration on Vβ8.2T cells in adult offspring ratsSecondary SEB administration had no impact on the percentages of Vβ8.2T cellsin the thymus of adult offspring rats in SEB group, but increased significantly thepercentages of Vβ8.2T cells in peripheral blood.4. Effect of SEB stimulation on in vitro cultured splenocytes in adult offspring ratsIt was found that the proliferation ability of in vitro cultured splenocytes to ConAor SEB stimulation was gradually increased with the increase of stimulated time.Furthermore, the proliferation ability of splenocytes to SEB stimulation was lower thanthat to ConA stimulation. The percentage of CD4+CD8T cells in cultured splenocyteswith SEB stimulation was significantly decreased in SEB group compared to ConAstimulation, but had no effect on the percentage of CD4CD8+T cells.5. Effect of maternal SEB administration during pregnancy on cytokines in theperipheral blood of adult offspring ratsThe levels of both IFN-γ and IL-4in the peripheral blood in SEB group weresignificantly increased compared to those in PBS group. When given SEB stimulationin adulthood again, the levels of both IFN-γ and IL-4(PBS+SEB, SEB+SEB) weresignificantly lower than those in their respective controls (PBS+PBS, SEB+PBS).6. Effect of maternal SEB administration during pregnancy on DNA methylation ofcytokines in the spleens of adult offspring ratsIt was found that maternal SEB administration during pregnancy significantlyreduced the methylation levels of both IL-4and IFN-γ gene in the spleens of adultoffspring rats. Conclusion:1. Maternal SEB administration during pregnancy changed the percentages of bothcentral and peripheral CD4/CD8T cells, and Vβ8.2T in adult offspring rats.2. Maternal SEB administration during pregnancy could change the immune response ofboth CD4/CD8T cells and Vβ8.2T cells to secondary SEB stimulation in adultoffspring rats.3. Maternal SEB administration during pregnancy decreased the proliferation ability ofin vitro cultured splenocytes to SEB stimulation in adult offspring rats. The decreasedproliferation ability could be associated with the decrease of CD4+T cellssubpopulation.4. Maternal SEB administration during pregnancy increased the levels of both IL-4andIFN-γ in peripheral blood of adult offspring rats, which could be resulted from thedecreased DNA methylation of both IL-4and IFN-γ genes.