Effect Of Endoplasmic Reticulum Stress Induced By Thapsigargin On Regulatory T Cell Function And Its Mechanism

Background: Regulatory T cells(Tregs)are a group of CD4+T lymphocyte subsets with significant immunoregulatory function.It plays a core regulatory role in the inflammatory response of sepsis by inhibite the function of immune cells.ER stress(ERS)is a state in which ER unfolded protein or misfolded protein aggregates under various physiological or pathological conditions,resulting in impaired ER function.The study found that ERS can regulate immune cell function and plays an important regulatory role in immune inflammation.Tregs play a crucial role in the inflammatory response of sepsis.It is not clear whether ERS affects the function of Tregs.Objective: Tregs were stimulated with ERS inducer thapsigargin(TG)or combined with LPS in vitro to explore the effect of ERS on Treg cell function and its mechanism.Methods: 1.Tregs and CD4 + T cells of normal BALB / C mice were isolated by immunomagnetic beads method.The purity of Tregs was identified by flow cytometry.Tregs were stimulated with low,medium and high doses of TG(0.05μM,0.1μM,0.2μM)in vitro for different time periods(6h,12 h,24h).Flow cytometry was used to detect the time and dose-response relationship of the forkhead helical transcription factor(Foxp3)and cytotoxic T cell-associated antigen 4(CTLA-4)expression.2.After TG stimulation,confocal microscopy was used to detect endoplasmic reticulum Western blotting,enzyme-linked immunosorbent assay(ELISA),CFSE tracer dye labeling used to detect the expression of GRP78,PERK,eIF2α,and ATF4 in Tregs,secretion of cytokines and T cell proliferation activity changes,respectively.Evaluate the functional status of Tregs.Combine PERK signaling pathway inhibitors(GSK2656157)to analyze the functional changes of Tregs after blocking the PERK signaling pathway,and to explore the molecular mechanism of ERS regulation of Treg function.3.LPS was used to induce sepsis in vitro.After TG was applied,the expressions of GRP78,PERK,eIF2α,ATF4,Foxp3 CTLA-4,cytokine secretion,Th1/Th2 differentiation and Teff were detected in the Treg cells using the above-mentioned experimental methods.Used GSK2656157 on Treg functional activity,analysis of the effects of ERS on the functional status of Treg and possible targets in sepsis in vitro.Result: 1.The purity of Treg and Teff were 91% and 88.2%,respectively.After TG(0.1μmol/L)stimulated for 12 h,Foxp3 expression was the most significant change,so TG 0.1μmol/L 12 h for subsequent experiments.But there is no significant change in the expression of Treg CTLA-4.2.After TG stimulation,confocal laser scanning microscopy revealed changes in the morphogenesis of Treg endoplasmic reticulum,endoplasmic reticulum fragmentation,swelling,and partial aggregation towards the nucleus;GRP78,PERK,ATF4,and eIF2α phosphorylation levels were significantly upregulated,while Treg Foxp3 Expression,IL-10 and TGF-β secretion were significantly increased,Teff co-cultured with Tregs,The proliferation activity was significantly inhibited,IL-4/IFN-γ ratio was significantly increased,IL-2 production decreased(P <0.05).After administration of GSK2656157,Foxp3 expression,IL-10 and TGF-β secretion were significantly reduced,Teff inhibited proliferation was inhibited,,and The ratio of IL-4/IFN.-γ decreased,IL-2 production increased(P<0.05).3.LPS-stimulated Treg mimics sepsis conditions in vitro.After TG stimulation,GRP78,PERK,ATF4 and eIF2α phosphorylation levels were significantly up-regulated.Treg Foxp3 expression,IL-10 and TGF-β secretion were significantly increased(P< 0.05),The proliferation of Teff in co-cultured with Tregs was significantly inhibited.The ratio of IL-4/IFN-γ was significantly increased and the production of IL-2 was decreased(P<0.05);After administration of GSK2656157,PERK,ATF4 and eIF2 alpha phosphorylation levels were significantly decreased,and Treg Foxp3 expression,IL-10 and TGF-β secretion were significantly reduced(P<0.05).The ratio of IL-4/IFN-γ was decreased,and the production of IL-2 was increased(P<0.05).Conclusion: ERS is a protective mechanism for cell response to internal and external environmental stimuli.Studies have shown that ERS has an important regulatory effect on the functional status of immune cells.The results of this study showed that:1.After administration of ERS inducer TG,the morphology of endoplasmic reticulum changed,the endoplasmic reticulum was disintegrated,expanded,and concentrated towards the side of the nucleus;phosphorylation levels of GRP78,PERK,ATF4,and eIF2α were significantly increased.,suggesting that TG induced ERS reaction,Treg Foxp3 expression,IL-10 and TGF-β secretion were significantly increased,Teff proliferation activity was significantly inhibited,culture supernatant IL-4/IFN-γ ratio increased significantly,IL The decreased production of-2 suggested that ERS induced by TG could significantly enhance the functional activity of Treg,but after application of PERK inhibitor,the functional activity of Treg was significantly reduced,suggesting that PERK signaling pathway is a possible target of Treg function.2.LPS-stimulated Treg simulating in vitro sepsis conditions,given TG stimulation,activation of the PERK signaling pathway to induce ERS response,while further enhancing the functional activity of Treg,and after the PERK inhibitor was given,by blocking the molecules of the PERK signaling pathway Expression,downregulation of Treg function.It is suggested that ERS may regulate the immune activity of sepsis through PERK signaling pathway and participate in the development of sepsis immune disorders.Blocking the Treg PERK signaling pathway may provide a potential target for the treatment of sepsis immune disorders...