The Role Of Transmembrane TNF-α-Mediated Reverse Signaling In Endotoxin Shock And Its Mechanism

TNF-α has two types:transmembrane TNF-α(tmTNF-α)and secretory TNF-α(sTNF-α).We and others have found that these two types of TNF-α play very different roles in endotoxic shock.sTNF-α participates in the occurrence and development of endotoxin shock as a pro-inflammatory factor,while tmTNF-α resists endotoxin shock through its anti-inflammatory effect.Transmembrane TNF-α can be used as a ligand to mediate the forward signal,but also as a receptor to transmit the reverse signal.Our previous work has confirmed the regulatory role of tmTNF-α forward signaling in endotoxic shock.However,the role of its reverse signaling in endotoxic shock is unclear.Based on previous studies,this study explored the role and mechanism of tmTNF-αreverse signaling in endotoxic shock through in vivo and in vitro experiments,and providing new ideas and experimental basis for clinical treatment of endotoxic shock.The main results are as follows:I.Transmembrane TNF-α reverse signaling inhibits LPS-induced inflammatory response1.Infliximab activated tmTNF-α reverse signaling inhibits the response of monocytes/macrophages to LPSInfliximab was added into THP-1 cells,mouse bone marrow-derived macrophages(BMDM)and human peripheral blood mononuclear cells for 30 minutes.After washing,they were stimulated with LPS.The results showed that infliximab can significantly inhibit LPS-induced IL-1β,IL-6,iNOS and IFN-β mRNA transcription and the secretion of these protein and NO,suggesting that infliximab activated tmTNF-αreverse signaling can inhibit the response of monocytes/macrophages to LPS.2.D4476 activated tmTNF-α reverse signaling inhibits the response of THP-1 cells to LPSSince Infliximab can neutralize sTNF-α and reduce LPS-induced inflammation,in order to rule out the influence of this factor,we pre-stimulated THP-1 cells with CKI inhibitor D4476 for 30 minutes to inhibit the phosphorylation of tmTNF-α and activate the reverse signaling of tmTNF-α,and then stimulated the THP-1 cells with LPS.The results showed that D4476 significantly reduced the mRNAlevels of IL-1β,IL-6,iNOS and IFN-β induced by LPS and the concentrations of IL-1β,IL-6,NO and IFN-β in the supernatant,but had no effect on IL-10.3.Infliximab activated tmTNF-α reverse signaling inhibits the response of TNFR1/R2KO BMDM to LPSTo further exclude the effect of tmTNF-α forward signaling and sTNF-α,we selected TNFR1/R2 knockout mouse derived BMDM,and pre-incubated these cells with infliximab for 30 minutes,and then stimulated with LPS.The results confirmed that Infliximab can significantly reduce LPS-induced IL-1β,IL-6,iNOS and IFN-βmRNA levels.The above results strongly suggest that tmTNF-α can suppress the response of monocytes/macrophages to LPS through its reverse signaling.Ⅱ.Molecular mechanisms of tmTNF-α reverse signaling inhibiting LPS induced inflammatory response1.Transmembrane TNF-α reverse signaling inhibits the activation of LPS/TLR4MyD88 signaling pathway1.1.Infliximab activated tmTNF-α reverse signaling inhibits LPS/TLR4-MyD88 signaling pathwayAfter incubated with infliximab for 30 minutes,THP-1 cells were stimulated with LPS for 0/15/30/60 minutes.Western blot results showed that infliximab can significantly inhibit LPS-induced degradation of IκB-α and phosphorylation of p65,and this effect increases with time.In addition,infliximab can also significantly reduce the phosphorylation levels of ERK1/2,JNK and p38,suggesting tmTNF-α reverse signaling can inhibit the activation of NF-κB and MAPK signaling pathway downstream of LPS/TLR4.1.2.D4476 activated tmTNF-α reverse signaling inhibits LPS/TLR4-MyD88 signaling pathwayTHP-1 cells were incubated with D4476 for 30 minutes,and then were stimulated with LPS for 1 hour.Western blot results showed that D4476 can inhibit LPS-induced IκB-α degradation and p65 phosphorylation,and reduce the phosphorylation levels of ERK1/2,JNK,and p38.2.The reverse signaling of tmTNF-α inhibits the activation of LPS/TLR4-TRIF signaling pathway2.1.Infliximab activated tmTNF-α reverse signaling inhibits LPS/TLR4-TRIF signaling pathwayWestern blot results showed that infliximab pretreatment of THP-1 cells for 30 min can significantly inhibit LPS-induced IRF3 phosphorylation,suggesting that infliximab-activated tmTNF-α reverse signaling can inhibit LPS/TLR4-TRIF signaling pathway.2.2.D4476 activated tmTNF-α reverse signaling inhibits LPS/TLR4-TRIF signaling pathwayTHP-1 cells were pretreated with D4476 to inhibit the phosphorylation of the cytoplasmic segment of tmTNF-α.Western blot results showed that D4476 also inhibited LPS-induced IRF3 phosphorylation,suggesting that the reverse signaling of tmTNF-α can inhibit the LPS/TLR4-TRIF signaling pathway.3.The reverse signaling of tmTNF-α promotes the expression of negative modulation molecule MCPIP1To explore the mechanism by which tmTNF-α reverse signaling inhibits the activation of TLR4 downstream signaling pathways,we used real time-PCR to detect the mRNA levels of MCPIP1,A20,SARM1,IRAK-M,rab7b,and CYLD downstream of TLR4,and the results showed that infliximab significantly promoted LPS-induced MCPIP1 mRNA transcription and protein synthesis,but has no effect on gene expression of other molecules.The above results suggest that the reverse signaling of tmTNF-α may play a role in resisting the LPS induced inflammatory responses by inducing the expression of the negative modulation molecule MCPIP1.III.Transmembrane TNF-α reverse signaling resists endotoxin shock of mice1.Transmembrane TNF-α reverse signaling resists endotoxin shock caused by LPS in mice1.1.Transmembrane TNF-α antibody enhances the resistance of infliximab in LPS-induced endotoxin shockWild-type BALB/c mice were injected with lethal LPS(30 mg/kg)intraperitoneally.In addition,a PBS control group,infliximab(30 mg/kg)and tmTNFα antibody(30 mg/kg)intervention group were set up,the results showed that the use of infliximab or tmTNF-α antibody alone can improve the survival rates of mice,from 5%to 20%or 23%,respectively,the combination of two antibodies can significantly improve the survival rate of mice to 60%.1.2.Transmembrane TNF-α antibody increases the expression of tmTNF-α and reduces the secretion of sTNF-α6 hours after LPS injection,it was found that infliximab does not affect the expression of tmTNF-α,but it can significantly reduce the concentration of serum sTNF-α,and the use of of tmTNF-α antibody and the combination of two antibodies can promote the expression of tmTNF-α,while reducing the concentration of serum sTNF-α.These results suggest that tmTNF-α antibody can promote the expression of tmTNF-α,enhance the reverse signaling of tmTNF-α activated by infliximab,and then play a synergistic role with infliximab.1.3.Infliximab and tmTNF-α antibody inhibits the production of endotoxin shock associated inflammatory factors6 hours and 12 hours after LPS injection,the serum inflammatory factors of mice were detected.The results confirmed that the single or combination of infliximab and tmTNF-α antibody can inhibit LPS-induced IL-1β,IL-6,IFN-β and NO produce.2.Transmembrane TNF-α antibody reduces the intervention dose of infliximab in endotoxin shock of miceSince tmTNF-α antibody can block the conversion of tmTNF-α to sTNF-α,that is,inhibit the production of sTNF-α.We reduced the dose of infliximab to 10 mg/kg,and found that although low-dose infliximab can still inhibit the above-mentioned LPSinduced release of inflammatory mediators such as IL-1β,IL-6,and NO,but has lost the protective effect on endotoxin shock mice,and no mice survived.However,when used in combination with tmTNF-α antibody,infliximab increased the survival rate of endotoxin shock mice to 60%.What is interesting is that whether the two antibodies were used alone or in combination,the effect of inhibiting inflammatory mediators is similar,but the combination of the two antibodies can significantly improve the survival rate of mice,suggesting that these two antibodies may also resist LPS induced endotoxin shock through other non-inflammatory mechanisms.3.Transmembrane TNF-α reverse signaling resists endotoxin shock of TNFR1/R2 knockout mice3.1.The reverse signaling of tmTNF-α improves the survival rate of endotoxin shock TNFR1/R2 knockout miceIn order to exclude the interference of the effects of tmTNF-α forward signaling and sTNF-α,we used TNFR1/R2 knockout mice to establish an endotoxin shock model.In addition,a PBS control group and an intervention group with infliximab(30 mg/kg)or tmTNF-α antibody(30 mg/kg)were given 30 minutes in advance.It was found that the deficiency of TNFR1 and TNFR2 can make the survival rate of mice after LPS stimulation reach 50%.Whether intervened with infliximab or tmTNF-α antibody alone,the survival rate of mice can be significantly increased to 83%and 92%,respectively.Because the neutralizing effect of infliximab on sTNF-α is removed due to the absence of TNFR,and tmTNF-α cannot transmit forward signaling without TNFR,the protective effects mentioned above can only be mediated by tmTNF-α reverse signaling activated by infliximab.3.2.Transmembrane TNF-α antibody promotes the expression of tmTNF-α and reduces the serum level of sTNF-α in TNFR1/R2 knockout mice6 hours after the LPS injection,the results of the two types of TNF-α detection were found that infliximab did not affect the expression of tmTNF-α,but significantly reduced the concentration of serum sTNF-α.Transmembrane TNF-α antibody can promote the expression of tmTNF-α,while reducing the concentration of serum sTNFα,suggesting that tmTNF-α antibody mainly inhibits the conversion of tmTNF-α to sTNF-α.Since there is no receptor,the effect of tm TNF-α antibody is mainly exerted through the reverse signaling of tm TNF-α.3.3.The reverse signaling of tmTNF-α inhibits the production of inflammatory factors in TNFR1/R2 knockout mice6 hours and 12 hours after LPS injection,the serum inflammatory factor detection results found that infliximab or tmTNF-α antibody can effectively inhibit LPS-induced IL-1β,IL-6,IFN-β and NO in TNFR1/R2 knockout mice.3.4.Transmembrane TNF-α reverse signaling inhibits the activation of TLR4 signaling pathway in the liver of TNFR1/R2 knockout mice6 hours after LPS injection,western blot were used to detect the signaling protein mentioned above in mouse liver tissues.The results showed that both infliximab and tmTNF-α antibodies could inhibit LPS-induced IκB-α degradation and ERK1/2 and p38 phosphorylation,suggesting that tmTNF-α reverse signaling can inhibit the activation of NF-κB and MAPK signaling pathways downstream of LPS/TLR4 in vivo.In summary,this study confirmed that tmTNF-α reverse signaling inhibits the activation of MyD88 and TRIF signaling pathways downstream of LPS-TLR4 by upregulating the negative regulated molecule MCPIP1,downregulates the secretion of pro-inflammatory cytokines,and promotes monocyte/macrophage resistance to LPS stimulation.And confirmed tmTNF-α reverse signaling can resist endotoxin shock in vivo,inhibit inflammatory response,and significantly improve the survival rate of mice.The combination of tmTNF-α antibody can enhance the reverse signaling of tmTNF-αactivated by infliximab and reduce the intervention dose of infliximab.The results of this study provide new experimental evidence and clues for the clinical treatment of endotoxin shock and inflammatory diseases.