The Role Of Transmembrane TNF-α-mediated Forward Signaling In Endotoxin Shock And Its Mechanism

Sepsis caused by infection is a systemic inflammatory response syndrome(SIRS),Excessive inflammatory response is thought to play a crucial role in the development of sepsis.TNF-α is an important inflammatory factor and exists as soluble TNF-α(sTNF-α)and transmembrane TNF-α(tmTNF-α).Several studies have demonstrated that sTNF-α plays an important role in endotoxic shock,and tmTNF-α may play a different role in endotoxic shock.tmTNF-α can act as a ligand to mediate the biological effects of target cells through forward signaling,or as a receptor to mediate the biological functions of tmTNF-α-expressing cells through reverse signaling.This study was to investigate the effect of forward signal on endotoxin shock and its molecular mechanism,and provide a theoretical basis for the application of tmTNF-αantibody in clinical shock.The main results are showed as follows:Ⅰ.Protective effect of tmTNF-α Ab on the different mortality of endotoxin shock1.The protective effect of tmTNF-α antibody on 100%lethal dose of endotoxin shock:As lethality was 100%,tmTNF-α polyclonal antibody(pAb)compared with IgG and commercialized TNF-α neutralizing antibody Infliximab,significantly improved the survival rate of LPS-induced endotoxin shock2.The protective effect of tmTNF-α antibody on 50%lethal dose of endotoxin shock:As lethality was 100%,both Infliximab and tmTNF-α Ab could improve the survival rate of endotoxin shock induced by LPS.But the effet of tmTNF-a Ab was superior to InfliximabⅡ.NIH3T3 cell high-expressing-tmTNF-α was obtainedNIH3T3 cells were infected with mouse wtTNF-α retrovirus,and the flow Cytometry results showed the expression of tmTNF-α on the surface of NIH3T3 cell membrane is about 70%,and the tmTNF-α expressing on NIH3T3 cell became a source of exogenous tmTNF-α to study its forward signalingⅢ.The effects of tmTNF-α forward signaling on the production of cytokines in LPS/TLR4 signaling pathway1.tmTNF-α forward signaling could inhibite IL-1β and IL-6 production of RAW264.7induced by LPS:The forward signaling of tmTNF-α inhibited LPS-induced IL-1β and IL-6 gene transcription in RAW264.7 cells,meanwhile reduced IL-1β and IL-6 secretion2.tmTNF-α forward signaling could also inhibite IL-1β and IL-6 production of BMDM induced by LPS:As the effect in RAW264.7,the forward signaling of tmTNF-α could also inhibite LPS-induced IL-1β and IL-6 gene transcription and protein secreation in BMDM3.tmTNF-α forward signaling could inhibite NO production induced by LPS in RAW264.7and BMDM:The tmTNF-α forward signaling could significantly inhibit LPS-induced iNO gene transcription and NO production4.The tmTNF-forward signaling had no effect on theproduction of IL-10:The tmTNF-αforward signaling had no effect on IL-10 gene transcription and protein secretionIV.The effect of tmTNF-a forward signaling on LPS/TLR4 signaling pathway1.The tmTNF-α forward signaling could inhibit the activation of NF-κB induced by LPS:After 30 min,IκB-α was significantly reduced.The amount of IKB-α in the LPS+Vector group was not different from that in the LPS group.While the LPS+tmTNF-α group significantly inhibited the degradation of IκB-α induced by LPS2.The tmTNF-α forward signaling could inhibit the activation of MAPK induced by LPS:After 30 min,the protein expression levels of p-ERK1/2,p-JNK1/2 and p-p38 were significantly increased.There was no significant difference between the LPS+Vector group and the LPS group.The LPS+tmTNF-α group significantly inhibited the increase of p-ERK1/2,p-JNK1/2 and p-p38 induced by LPS stimulation V.Exogenous tmTNF-α exerted anti-inflammatory effect through TNFR21.TNFR1 had no effect on the effect of tmTNF-α forward signaling:The results of ELISA,RT-PCR and Western Blot showed that the tmTNF-α forward signaling could still inhibite the inflammation in BMDM from TNFR1 knock out mouse2.TNFR2 gene knockout eliminated the anti-inflammationary effect of tmTNF-α forward signaling:ELISA,RT-PCR and Western Blot showed that the tmTNF-α forward signaling had no influence on the reaction to LPS on BMDM from TNFR2 knock out mouseVI.The effect of tmTNF-α forward signaling on the negative regulatory molecules of LPS/TLR4 pathway1.The effect of tmTNF-α forward signaling on the mRNA level of negative regulatory molecules in the LPS/TLR4 pathway:After 4 hours of LPS stimulation,the mRNA expressions of A20,MCPIP1 and Triad3A in LPS+tmTNF-α group were significantly higher than those in LPS+Vector group and LPS group.There was no significant difference between LPS+Vector group and LPS group2.The effect of tmTNF-α forward signaling on MCPIP1 protein expression:Western blot showed that MCPIP1 expression was significantly increased after LPS stimulation for 3 h,and MCPIP1 were higher in LPS+tmTNF-α group than in LPS+Vector group3.siMCPIPl partially abolished the inhibitory effect of tmTNF-α forward signaling:RT-PCR results showed that si MCPIP1 partially restored IL-1p and IL-6 gene expression in LPS+tmTNF-α groupⅦ.Effects of tmTNF-α Ab on lethality and inflammatory factors in endotoxin shock of WT and TNFR2 knockout mice1.The effect of tmTNF-α Ab on the survival rate of endotoxin shock of WT and TNFR2 knockout mice:In LPS+IgG groups,the survival rate of TNFR2 knock out mice was higher than that in WT mice.Meanwhile In LPS+tmTNF-α Ab groups,the survival rate of TNFR2 knock out mice was significantly higher than that in WT mice2.The effect of tmTNF-α Ab on the tmTNF-α expression and inflammatory factors in endotoxin shock of WT and TNFR2 knockout mice:tmTNF-α Ab could increase the expression of tmTNF-α in both kind of mice,and significantly inhibited the secretion of serum TNF-α,IL-1β,IL-6 concentrationConclusion:tmTNF-α can act on macrophages through TNFR2,inhibiting the response of macrophages to LPS and attenuating inflammatory response.tmTNF-αcould directly act on the LPS/TLR4 signaling pathway to inhibit the activation of NF-κB and MAPK,and could also inhibit inflammation by more expression of some negative regulatory molecules.In vivo,the role of tmTNF-α forward signaling also confirmed.Due to loss of forward signaling in TNFR2 knockout mice,there was much lower decreation of IL-1β and IL-6 than in WT mice.