Interferon-γ Regulates Immunosuppression In Septic Mice By Promoting The Warburg Effect Through The PI3K/Akt/mTOR Pathway

Objective: The main cause of high mortality in sepsis is organ dysfunction due to immunosuppression,and reversing sepsis immunosuppression is the key to sepsis treatment.It has now been found that the Warburg effect occurs during immune cell activation.The Warburg effect was first found in tumor cells,which usually exhibit an intensified metabolism,in other words,tumor cells tend to supply energy in a glycolytic manner,i.e.,the Warburg effect,even under conditions of adequate oxygenation.During the onset of sepsis,immune cell metabolism shifts to the Warburg effect,whereas during the immunosuppressive phase of sepsis,the Warburg effect is suppressed.Interferon gamma(IFNg)is a potential therapy for immunosuppression in sepsis,promoting glycolysis to restore the metabolic defect in monocytes,but the therapeutic mechanism is not known.Therefore,it is hypothesized that IFNg regulates sepsis immunosuppression by promoting the Warburg effect.In this study,2-DG was intervened with LY294002 by in vivo in vitro experiments.IFNg was investigated to promote Warburg effect through PI3K/Akt/m TOR pathway to regulate immune function in septic mice.Research method: In this experiment,8-week-old male C57BL/6 mice and DC2.4mice with dendritic cells were used as experimental subjects.Two in vitro and in vivo sepsis models were established using CLP-treated C57BL/6 mice and LPS-stimulated DC2.4,respectively,and IFNg was given as the therapeutic drug.In vivo study:SPF-grade healthy male C57BL/6 mice were randomly divided into sham-operated group,sepsis group,IFNg+sham-operated group,IFNg+sepsis group,2-DG+sham-operated group,2-DG+sepsis group,and IFNg+2-DG+sepsis group.The mouse sepsis model was constructed by CLP surgery,and the sham-operated group was given the same open surgery.IFNg(0.01 μg/g)was injected into the tail vein 30 min before model construction for therapeutic intervention,and 2-DG(500 mg/kg)was injected intraperitoneally 3 h before for Warburg effect inhibition intervention.24 h later,mice were executed and peripheral blood and spleen were taken.The spleen weight mg/mouse body weight g(splenic index)was weighed to roughly assess the immune function of mice,Simon score to evaluate the severity grade of abdominal infection,HE staining to assess the pathological score to evaluate the degree of spleen damage,immunohistochemistry to detect the expression of HLA-DR and Caspase-3in the spleen to assess the spleen function,extraction of primary spleen cells,flow cytometry to detect CD4+CD25+Foxp3+ Treg percentage,DC surface CD86 and PD-L1 expression.ELISA to detect TNF-α,IL-6 and IL-10 secretion levels in peripheral blood and LPS-stimulated primary cell supernatant in mice.Biochemical kits were used to detect glucose uptake,extracellular lactate production,and intracellular ATP content in mouse splenocytes.western-blot assay detected the protein expression levels of Glut1,Glut4,LDH,PDH,PDK1,PI3 K,p-PI3 K,Akt,p-Akt,m TOR,p-m TOR,and HIF-1α,responding to the Warburg effect level with PI3K/Akt/m TOR pathway.In vitro study:DC2.4 mouse dendritic cells were grouped in the same in vivo study and were randomized into normal control group,sepsis group(LPS stimulated),IFNg group,IFNg+sepsis group,2-DG group,2-DG+sepsis group,IFNg+2-DG+sepsis group,LY294002+IFNg+sepsis group.DC2.4 cells were stimulated by 5ug/ml of LPS for 24 hours to simulate sepsis,treated with 20ng/ml IFNg and 25 m M 2-DG with10 m M LY294002 intervention was administered.After 24 hours of drug action,cells as well as supernatants were collected.CCK-8 was used to detect cell viability,PT-PCR to assess m RNA levels of Bcl-2 and Bax,Tunel to detect apoptosis,laser confocal to detect LC3B-labeled autophagic vesicles,and biochemical kits to detect glucose uptake,extracellular lactate production,and intracellular ATP content in dendritic cells.-blot assay detected the protein expression levels of Glut1,Glut4,LDH,PDH,PDK1,PI3 K,p-PI3 K,Akt,p-Akt,m TOR,p-m TOR,HIF-1α,responding to the Warburg effect level and PI3K/Akt/m TOR pathway.Results: First,in vivo experiments revealed that the spleen and immune function of septic mice were severely impaired,with reduced splenic index and HLA-DR expression,and increased expression of Simon index,pathology score and Caspase-3.The flow assay of primary splenocytes showed that the percentage of Treg was increased,the expression of CD86 on the surface of DCs was decreased,and the expression of PD-L1 was increased in the splenocytes of septic mice.ELISA showed that TNF-α,IL-6 and IL-10 were increased in peripheral blood,and the secretion capacity of TNF-α and IL-6 was decreased and the secretion of negative regulator IL-10 was increased in the supernatant of primary cells.Warburg effect-related indicators(glucose uptake,lactate production,ATP content with Glut1,Glut4,LDH,PDH)were decreased.PI3K/Akt/m TOR/HIF-1α pathway was inhibited.IFNg treatment suppressed sepsis-induced splenic damage and immunosuppression,and also increased the Warburg effect and PI3K/Akt/m TOR/HIF-1α pathway levels.2-DG intervention suppressed the Warburg effect and the therapeutic effect of IFNg.In vitro experiments revealed that LPS-induced sepsis resulted in decreased DC cell viability,elevated apoptotic gene Bax,decreased apoptosis-protective gene Bcl-2,elevated LC3 B expression,and elevated apoptosis detected by Tunel.As well as reduced expression of indicators related to Warburg effect,PI3K/Akt/m TOR/HIF-1αpathway was inhibited.IFNg treatment inhibited sepsis-induced apoptosis and autophagy and restored cell viability and Warburg effect.2-DG intervention to inhibit Warburg effect also inhibited the therapeutic effect of IFNg.The therapeutic effect of IFNg was also inhibited after LY294002 intervention inhibited PI3 K pathway.Conclusion: In summary,our findings found that IFNg improves immunosuppression in mice with sepsis,along with an enhancement of the Warburg effect,by activating the PI3K/Akt/m TOR/HIF-1α signaling pathway.Therefore,our study elucidates the molecular signaling mechanisms associated with sepsis immunosuppression,and the Akt-m TOR-HIF-1α signaling pathway and glycolytic-related enzymes such as hexokinase are targets for intervention in the Warburg effect,and these targets provide new ideas for immunotherapy of sepsis.