| CD4+T helper cells (Th cells) is a key component in adaptive immune system. The type of the Th cells strongly influences the outcome of the immune responses. In normal adult condition, the differentiation and regulation of Th subsets are well-organized to maintain immune homeostasis, but in pathologic conditions, the balance is broken with the significant decrease of ratio of Treg/Th17and Treg/Th1cells, these alterations of CD4+T cell subsets are significantly correlated with metabolic disorders. advanced glycation end products(AGEs) are a group of heterogeneous molecules formed by non-oxidative and oxidative reactions of sugars with proteins and/lipids, Involved in the occurrence and development of a variety of immune related diseases. However, little attention has been paid to the effects of AGEs on CD4+T cell differentiation. In this study, human CD4+T cells were treated with AGE to evaluate effects of AGEs on CD4+T cell phenotype transition and Treg suppressive function, to explore the underlying mechanisms, and to provide new ideas for clinical treatment of related diseases from the perspective of intervention Th cell differentiation.Part ⅠEffects of AGEs on CD4+T cell differentiationObjective:To explore the effects of AGE on CD4+T cell differentiation. Methods:To determine the effect of AGEs on naive CD4+T cell differentiation, fresh isolated naive CD4+T cells were treated with AGEs for72h At the meantime, cells treated with BSA (50mg/ml), Glucose (2.5M) or completed T cell culture medium (CTCM) were used as control. After72h incubation, the cells phenotypes were detected by surface and intracellular cytokine staining by Flow cytometry. Total RNA were extracted from treated cells and used to detect Th-type specific transcription factors by realtime PCR. The naive CD4+T cells were co-cultured with or without Treg cells(naive:Treg=4:1) for56h prior to addition of [3H]-thymidine for another16h, the resulting cells were detected by liquid scintillation counter. Results:Compared with CTCM, BSA and glucose, AGEs obviously increased IFNy positive cell percentage in CD4gate (p<0.05, respectively, and no significant differences were found in IL-4and FoxP3positive cells percentage in CD4gate among AGEs and other groups. Realtime PCR data demonstrated the gene levels of Tbet and RORyt were higher in naive CD4+T cells treated with AGEs than with CTCM, BSA or glucose (p<0.05, respectively), however there were no significant difference in the gene expression levels of GATA3and FoxP3among AGEs and other treated cells. The naive CD4+T cells were co-cultured with or without Treg cells. Liquid scintillation counter data indicated that the naive CD4+T cells proliferation was decreased by86.89%in co-culture with Treg cells only, whereas the suppressive capacity of Treg cells was effectively eliminated by the addition of AGEs in a dose dependent manner. Conclusion: AGEs induced naive CD4+T cells transition into Thl and Th17cell types and reversed the suppressive function of Treg cells, which promoted pro-inflammatory developments.Part Ⅱ The role of RAGE in AGEs-induced CD4+T cell development into pro-inflammatory responseObjective: To explore the role of RAGE in AGEs-induced CD4+T cell development into pro-inflammatory response. Methods:Freshly isolated naive CD4+T cells were treated with AGEs or BSA or high glucose for24h, whole cell extracts and total RNA were prepared. Protein and geneexpression of RAGE were detected by Western blotting analysis and realtime PCR respectively. IFNy and IL-17a were determined by flow cytometry after the RAGE gene was slienced in naive CD4+T cells by shRNA. After cocultured with RAGE shRNA silenced Treg cells, The CD4+T cells were collected to detect the proliferation by liquid scintillation counter. Results: Compare with untreated cells transduced with no shRNA, or with the Luc shRNA, naive CD4+T cells transduced with RAGE shRNA showed significantly lower percentage of INFγ(p=0.003) or IL-17A(p=0.004)positive cells. Treg cells not treated by shRNA apparently inhibited naive CD4+T cell proliferation,and AGEs reversed the inhibitory effect of Treg cells. However, AGEs had no capability to ameliorate the proliferation inhibition of naive CD4+T cells induced by Treg cells transduced with RAGE shRNA. Conclusion:RAGE mediated the cells transition from naive CD4+T cells to Thl and Th17induced by AGEs. And the elimination of Treg cells suppressive function by AGE was also executed through AGEs-RAGE axis. Part III The role of PPARy in AGEs-induced CD4+T cell development into pro-inflammatory responseObjective: To explore the role of PPARy in AGEs-induced CD4+T cell developm-ent into pro-inflammatory response. Methods: PPARy gene expressions were detected by Western blotting analysis in naive CD4+T cells transduced with or without RAGE shRNA prior to incubation with AGEs. Naive CD4+T cells were treated with PGJ2, a natural PPARy agonist before incubation with AGE, IFNy or IL-17a positive cells were detected by flow cytometry. RAGE shRNA transduced naive CD4+T cells were treated with PD68235, and the percentage AGE of IFNy or IL-17a positive cells were determined. Freshly isolated Treg cells transduced with or without RAGE shRNA were treated with PGJ2or PD68235before incubation with AGEs, then co-cultured with naive CD4+T cells prior to addition of [3H]-thymidine. The CD4+T cell proliferation was analysed. Results:Western blotting analysis showed that, compared with control, PPARy gene expression was reduced in naive CD4+T cells treated with AGEs only, whereas the gene expression was unaffected in cells transduced with RAGE shRNA before incubation with AGEs. AGEs increased the percentage of IFNγ or IL-17A positive cells in the gate of CD4cells(2nd column), but PGJ2obviously attenuated the effects. The percentage AGE of IFNy or IL-17a positive cells returned higher in RAGE shRNA transduced naive CD4+T cells treated by PD68235than in those cells not treated (p=0.003). PGJ2reversed the effect of AGEs on reduction of the suppressive function of Treg cells, whereas PD68235abrogated the suppressive function of Treg cells. Conclusion:The elimination of PPARy activity was essential for AGEs in inducing naive CD4+T cell differentiation as well as reducing the suppressive function of Treg cells. |
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