Effect And Mechanism Of Mesenchymal Stem Cells On The Senescence Of CD4+T Cells In Systemic Lupus Erythematosus

Background:Excessive T-cell proliferation with the subsequent clonal expansion is common immune imbalance phenomenon during the progression of systemic lupus erythematosus(SLE).As such,senescence of hyperactive T cells,accompanied with cell cycle arrest,could potentially reduce systemic inflammation and ameliorate SLE symptoms.Although human umbilical cord-derived mesenchymal stem cells(hUC-MSCs)transplantation has been proved to be an effective therapeutic approach to treat SLE,the function and detailed underlying mechanisms in senescence of T cells are not clear yet.Objectives:To examine the specific effects and the molecular mechanism of hUC-MSCs on CD4+T cell senescence in SLE,to reveal the new theoretical basis for hUC-MSCs transplantation.Methods:hUC-MSCs were injected into MRL/lpr lupus mice via the tail vein to observe the effect and mechanism of hUC-MSCs on the phenotype of lupus disease and cell senescence.Magnetic beads were used to sort splenic CD4+T cells and co-cultured with hUC-MSCs in direct contact or a transwell system to further clarify the effect and mechanism of hUC-MSCs on T cell senescence.The splenic CD4+T cells were treated with Sirtl activator and inhibitor respectively,and co-cultured with hUC-MSCs in vitro to explore whether Sirtl is a mediator of hUC-MSCs increasing CD4+T cell senescence.Real-time fluorescent quantitative polymerase chain reaction(real time PCR)was used to screen miRNAs that may target Sirt1.We also designed luciferase experiment to provide evidence of the direct interaction between the miRNA and Sirtl mRNA.In addition,CD4+T cells were co-cultured with miR-199a-5p mimic or inhibitor to observe the effects of miRNA on CD4+T cell senescence and related signal pathways.MRL/lpr splenic CD4+T cells and hUC-MSCs were cultured alone or together in the presence or absence of RNase using a transwell system,to determine whether hUC-MSCs is the source of miRNA.Furthermore,miR-199a-5p agomir or micrONTM agomir Negative Control were respectively infused into MRL/lpr mice for assessing their effects on splenic CD4+T cell senescence and lupus symptoms in vivo.Results:We showed that hUC-MSCs transplantation ameliorated lupus symptoms and increased senescence of splenic CD4+T cells through Sirtl/p53 signaling in MRL/lpr mice.hUC-MSCs could reduce Sirtl level and increase CD4+T cell senescence in both cell-cell contact and a transwell system in vitro.Moreover,Sirtl was a key mediator whereby hUC-MSCs increase senescence of splenic CD4+T cells.qPCR analysis showed that of these 10 miRNAs,only miR-199a-5p was significantly decreased in MRL/lpr splenic CD4+T cells and increased after hUC-MSCs infusion.Dual luciferase experiment showed the direct interaction between the miR-199a-5p and Sirtl mRNA.MiR-199a-5p mimic reduced the expression of Sirtl in MRL/lpr CD4+T cells and increased cell senescence and miR-199a-5p inhibitor increased the expression of Sirtl in wild-type CD4+T cells and reduced cell senescence in vitro.The expression of miR-199a-5p in hUC-MSCs was significantly increased compared with splenic CD4+T cells of MRL/lpr mice.Co-culture of hUC-MSCs with MRL/lpr splenic CD4+T cells in a transwell system for 48 h showed increased intracellular miR-199a-5p along with decreased Sirtl gene expression and restored expression levels of senescence markers in the CD4+T cells,while miR-199a-5p inhibitor treatment remained depressed status.MRL/lpr splenic CD4+T cells and hUC-MSCs were cultured alone or together with and without RNase using a transwell system,RNase significantly reduced the level of miR-199a-5p in the hUC-MSCs culture supernatant and inhibited the effect of hUC-MSCs on CD4+T cells senescence.In addition,systemic delivery of miR-199a-5p agomir in MRL/lpr mice decreased serum levels of anti-dsDNA antibody and IgG.Renal impairments were also ameliorated in the miR-199a-5p agomir treated group,as shown by reduced glomerular enlargement and hyper cellularity and less IgG deposition in the peripheral capillary loops.However,CD4+T cell senescence was increased through Sirtl,mimicking the therapeutic effects of transplanted hUC-MSCs.Conclusion:We have shown here that hUC-MSCs increase splenic CD4+T cell senescence through the Sirtl/p53 pathway via miR-199a-5p in lupus-prone MRL/lpr mice.These findings not only extend our knowledge of the immunoregulatory function of hUC-MSCs in SLE,but also offer miR-199a-5p as a potential biomarker or therapeutic target to further explore in the future.