Anti-infection Effect And Mechanism Of Thymic Epithelial Cell Conditional Knockout Of GSK-3β Gene On Aging Mice

Background China is rapidly entering a moderately aging society.With age after puberty,the thymus shrinks and degenerates,and the output of mature T cells decreases,which increases the risk of infection,tumors,and autoimmune diseases among the elderly.Therefore,it is very important to explore thymus reconstruction and T cell regeneration.Objective Observing thymic epithelial cells（thymic epithelial cells,TECs）conditional knockout of GSK-3βgene aging mice against Listeria monocytogenes(ovalbumin _257-264-expressing Listeria monocytogenes,Lm-OVA)infection carrying chicken ovalbumin short peptide effect,and to explore the corresponding mechanism,to provide experimental basis for individuals in clinical aging to enhance immunity against infection.Methods（1）Pair the mice GSK-3β-floxp and FoxN1-Cre transgenic mice to obtain TECs-deficient GSK-3βmice（cKO group）and raise them with littermate wild-type（WT group）mice up to 36 weeks old.（2）Infection with Lm-OVA for the first time and second time（interval 4weeks）respectively.After 3 days,collect peripheral blood and detect biochemical indicators to clarify liver and kidney toxicity,then kill the mice by euthanasia and obtain thymus,spleen and liver and kidney tissues,measure the size and weight of thymus and spleen in mice,prepare thymus by mechanical method,and digest with enzymes The thymocyte suspension of TECs was prepared by the method,the thymus was counted,and the thymocytes and TECs subpopulations were detected by FCM.（3）The liver gallbladder lobe and the right kidney of the mice in the WT group and the cKO group were homogenized and smeared on the tryptone soy agar plate,and the typical Lm-OVA colonies were counted after 24 hours.（4）Take the spleen in（2）and prepare a single suspension cell of the spleen.One part is used for FCM to detect the changes of spleen T cell subsets;the other part is co-cultured with heat-inactivated Lm-OVA,and FCM is used to detect spleen T cell subsets,specific CD8⁺T cells and cytokines and key factors secreted by T cells protein expression.Results（1）The growth and development of the 40 weeks old cKO mice were normal,and the thymus volume was increased compared with that of the WT group,without obvious appearance and liver and kidney toxicity,the thymocyte subgroups,TECs subgroups and spleen T cell subgroups were all normal.There is a clear upward trend.（2）Compared with the WT group,the T_CM and T_EM of cKO mice showed a significant increase.（3）When Lm-OVA was co-cultured with spleen T cells,the expression of specific CD8 T cells was enhanced.（4）The positive factors TNF and IFN-γsecreted by T cells increased,the negative factors TGF-β1 and IL-10 decreased.（5）The expression of anti-CD8 T cell apoptosis molecule Bcl-2 on T cells increased,the expression of anti-T cell activation molecule PD-1 decreased.Conclusion Conditional knockout of GSK-3βin TECs promoted the expression of downstream related genes,thereby promoting the proliferation of TECs and enhancing the function of TECs,which continued to promote the regeneration of T cells.After infection with Lm-OVA,the anti-Lm-OVA-specific CD8⁺T cells and CD8⁺SLEC cells in cKO mice increased,and the ability of these cells to secrete anti-infection cytokines was enhanced,while the secretion of cytokines that suppressed immune responses was reduced.At the same time,the anti-apoptotic ability of T cells is enhanced,and it is easier to activate.Further,the expression of PD-1,an inhibitory molecule of T cell activation,is reduced,so that T cells have stronger anti-infection ability.