The Effect And Mechanism Of Tumor Necrosis Factor-α Induced Protein 8 Like-2 In Regulating The Endoplasmic Reticulum Stress In Dendritic Cells Under Septic Challenge

BACKGROUND AND OBJECTIVE:Tumor necrosis factor-α induced protein 8 like-2(TIPE2)is a cytoplasmic protein preferential expressed in immune cells.It functions as a negative immune regulator,which can counteract excessive inflammatory response and play a pivotal role in maintaining balance between innate and acquired immunity.In our preliminary studies,we demonstrated that TIPE2 is abundant in quiescent dendritic cells and can attenuate hyperactivated immunity of splenic dendritic cells,consequently improve the survival of severely scalded mice.However,the precise mechanism underlying the protective effect of TIPE2 is yet to be further defined.On the other hand,accumulating evidence has reported the unique and crucial role of ERS upon the immunity of dendritic cell.Herein,the present study aimed to detect whether TIPE2 can protect dendritic cells immune function via down-regulating aberrant ERS in the setting of septic challenge.METHODS:Method 1.To eatablish a moderate mouse sepsis model(CLP),and extract DC from septic mice spleens with magnetic bead method.Immunofluorescence laser confocal technology was used to detect the levels of GRP78(ERS marker)and TIPE2 in DC,and Western blot was used to detect the changes of GRP78 and TIPE2 protein levels;4-PBA(one of endoplasmic reticulum stress pan inhibitor)was used preadministration to investigate its effect on GRP78 and TIPE2 levels in septic DC.2.Replicate tipe2 gene knockout mice(TIPE2-KO)and tipe2 gene overexpression mice(TIPE2-KI)sepsis models,observe mice survival rate;flow cytometry estimates DC surface molecules and cell apoptosis changes;Western blot detects ERS-related protein levels;ELISA method tests TNF-α,IL-6,IL-12levels;CCK-8 method examines the effects of DC on T cells proliferation after mixed lymphocyte response;3.According to the results of the foregoing experiments,in vivo and in vitro experiments(endotoxin stress to simulate sepsis),specific ERS signal pathway inhibitor was use to observe its effects on TIPE2-KO DC ERS-related signal proteins and cell apoptosis,as well as the effect on the damage of important organs and survival rate of TIPE2-KO sepsis mice.4.In vivo experiment,the signal pathway of TIPE2 regulating ERS was initially explored.5.Estimate the effect of TIPE2-KI DC adoptive transferring on the cerebral ERS and inflammation in the brain of septic aged mice after sevoflurane exposure.STATISTICAL ANALYSIS:Data were represented as mean ± standard deviation(SD)and analyzed by SPSS 25.0statistical analysis software.Student’t test was used to test significant differences of intergroup,and one-way analysis of variance(One-Way ANOVA)was perform for assessing significant differences among the groups.Kaplan-Meier statistical method and log-rank test were used to calculate and analyze the survival rate of septic mice.P<0.05 means significant statistic.RESUTS AND CONCLUSIONS:1.Confocal laser microscope imaging analysis showed that as sepsis progressed,GRP78 fluorescence in mice DC gradually increased and TIPE2 fluorescence gradually decreased.Western blot results showed that GRP78 and TIPE2 protein levels were negatively correlated.Furthermore,4-PBA(Endoplasmic reticulum stress inhibitor)intraperitoneal preadministration can alleviate this effect.2.Flow cytometry results showed that under septic stress,CD80 and CD86 were highly expressed in TIPE2-KO DC accompanying with enhanced cells apoptosis.The above effects were suppressed in TIPE2-KI septic DC.3.Western blot results showed that GRP78 level reach the peak in TIPE2-KO septic DC,and was the lowest in TIPE2-KI septic DC.Further analysis revealed that the activity of IRE1α,one of ERS key mediator,was hyperactivated in TIPE2-KO DC.4.ELISA results showed that production of TNF-α,IL-6,and IL-12 in TIPE2-KO DC were enhanced,accompanying with aggravated proliferation of T cells after mixed lymphocytes incubation.5.Pretreatment with IRE1α inhibitor(4u8c)can attenuate phenotype,cells apoptosis,and inflammatory cytokine production in TIPE2-KO septic DC.6.TIPE2 can mitigate the endoplasmic reticulum stress and inflammation levels in mice brain.6.In vitro experiment,4u8 c can alleviate hyperactivitation of IRE1 α and reduce cell apoptosis in endotoxin-stressed TIPE2-KO DC.7.Further study found that,during sepsis,the activity of m TOR was exclusively enhaced and one downstream target of m TOR-p70S6 K has hyperractivity in TIPE2-KO DC.Furthermore,inhibition of m TOR activity can modulate excessive IRE1 α phosphorylation and rescue TIPE2-KO DC from septic apoptosis.8.TIPE2 KI DC adoptive transferring via tail vein can ameliorate the enhanced endoplasmic reticulum stress level and the accompanying inflammation in the brains of septic aged mice after sevoflurane exposure.Conclusions: TIPE2 can significantly ameliorate aberrant immunoreactivity of dendritic cells in septic mice,which may be related to its down-regulation of m TOR activity and subsequently mitigate hyperactivation of IRE1αpathway among the endoplasmic reticulum stress response.