Targeted Inhibition Of RSK To Regulate The Development Of Thymic Epithelial Cells And Promote The Proliferation Of T Cells In Aged Mice

Background The aging of the Chinese society’s population has been increasing year by year.With the growth of age,the thymus is one of the earliest organs to decline.The decline of the thymus leads to the decrease of the output of T cells,which is also an important reason for the weakened immunity of aged individuals.How to promote thymus reconstitution and T cells regeneration,so as to enhance the immunity of aged individuals has become a current research hotspot.Objective Observe the effects of targeted inhibition of RSK on promoting the development of mouse thymic epithelial cells and T cell regeneration in aging and aged mice,and explore the corresponding molecular mechanisms,providing an experimental basis for enhancing the immunity of aged individuals clinically.Methods Firstly,the expressions of RSK1,RSK2,RSK3,RSK4 in the TECs of1-month-old mice and 3-month-old mice were detected by Flow Cytometry(FCM),respectively.C57BL/6J mice were raised to the aged stage(20-month-old),and were randomly divided into the control group and the experimental group.The experimental group was intraperitoneally injected with 0.5 mg/Kg of BI-D1870,an inhibitor of RSK,every other day,and the control group was injected the same as BI-D1870 solvent,PBS solution containing an equal volume of DMSO(the following were referred to as the DMSO group),the mice were raised for one more week after the administration for 7 times.Then the mice were sacrificed.The changes of the structure of thymic cortical and medullary of the aged mice were evaluated by immunofluorescence technique.Thymocytes,TECs and splenocytes were prepared and counted.The number and proportion of thymocytes and their subsets,TECs and their subsets,T cells and their subsets in the spleen of aged mice were detected by FCM.The liver,kidney tissues and peripheral blood of mice were harvested to assess the side effects induced by BI-D1870 probably with HE staining and Biochemical index detection,respectively.The ageing mice(more than 4-month-old)were randomly divided into control group and experimental group.The process modes and test indexes by FCM were the same as those in aged mice above.Reverse transcription-quantitative polymerase chain reactin was employed for examining changes in the expression of genes related to the development and function of TECs in aged mice.In vitro experiments,the determined concentration and effective acting time of the doxorubicin(DOX)that induces senescence were applied to treat the thymic epithelial cell line iTEC.The expression of RSK1,RSK2,RSK3,and RSK4 before and after inducing iTEC senescence was detected by FCM.Furthermore,the effect of siRNA-RSK or RSK3 overexpression plasmid(GV362-RSK3)transfected into senescent iTEC had been verified.The changes on the expressions of Ki-67,CD80 and MHC II of senescent iTEC cells in different treatment groups were detected by FCM.Western blot was hired to measure the changes on the expression of proteins located in upstream and downstream of MAPK/ERK signaling in the different treatment group.Results All RSK isoforms were expressed in TECs in 3 month-old mice,among the four different isoforms,RSK3 expression showed the highest level,and RSK4 expression level was the lowest.Compared with young mice,the expression of RSK isoforms increased significantly,among which RSK3 was increased the most,followed by RSK1 and RSK2 in the adult mice.BI-D1870 treatment made the cortical and medullary structure of the thymus in aged mice clear and heightened the numbers of all thymocytes subgroups,TECs and their subpopulations,and T cells and their subpopulations in the spleen of aged and aging mice.Furthermore,BI-D1870 facilitated the expression of genes containing FOXN1、AIRE、Csna、DLL4、CXCL12、Cts L、Fgf R2 IIIb which are related to TECs development and function.In vitro experiments,the expression of RSK isoforms was increased in senescent iTEC.After silencing the three isoforms of RSK or overexpressing RSK3 in senescent iTEC cells,the expression of RSK in senescent iTEC decreased or increased.After BI-D1870 treatment or siRNA silencing RSK gene,the expressions of Ki-67,CD80 and MHC II in senescent iTEC cells were enhanced.Likewise,the express levels of phosphorylated p-70S6 K and phosphorylated S6 protein in the senescent iTEC cell line were boosted.Conclusion The expressions of four isoforms of RSK in TECs increase significantly during the aging process.Targeted inhibition of RSK can reduce the excessively high activity of RSK in aged TECs to an appropriate level,subsequently up-regulating the expression of downstream phosphorylated p70S6 K and phosphorylated S6 protein,promoting the number of TECs in both aging and aged mice and delaying the aging process of the cortical and medullary of mice,heightening the number of thymocytes and subgroups,increasing naive T cells in the spleen,which is induced by the enhanced function-thymus.Therefore,targeted inhibition of RSK can postpone the thymic aging process and promote the reconstructing of the thymus and the regeneration of T cells in aged mice.This experiment provides an experimental basis for enhancing the immunity of aged individuals through thymus reconstitution in clinical practice.