4-Octyl Itaconate As A Metabolite Derivative Inhibits Inflammation Via Alkylation Of STING

Objective:STING is an important immune molecule in innate immunity and plays an important role in the occurrence and development of sepsis.Inhibiting the activation of STING is an important regulatory target.Itaconate is an anti-inflammatory metabolite,produced by the Irg1-encoded enzyme.the anti-inflammatory mechanism of the IRG1-Itaconate axis is not clear as a regulatory hub for macrophage immunity and metabolism.1.To study how STING signaling pathway stimulates the generation of IRG1-itaconate.2.How 4-octyl itaconate(4-OI)as a metabolite derivative regulates STING signaling pathway.3.Effects of itaconate and its derivative 4-OI on sepsis in mice.Research methods:1.Metabolomics was performed after STING agonist was used in RAW264.7 for 6h and 16 h to observe the changes of metabolites.By using STING KO,TBK1 KO RAW264.7,IFN-AR KO BMDM and IRF3 small interference,the experiment reveals how STING stimulates the production of IRG1-itaconate.2.RAW264.7,THP1 and BMDM cells were pretreated with itaconate and 4-OI,and stimulated with STING agonist DMXAA or 2 ’3’ c GAMP for 1h,to observe the changes in the phosphorylation of STING and its downstream signal molecules and the expression levels of inflammatory factors.HEK293 T cells overexpressed STING protein,itaconate or 4-OI pretreatment.Mass spectrometry(LC-MS)was used to detect whether 4-OI had alkylation modification on STING cysteine and the modification site.The mutant plasmid was constructed from STING modified cysteine and transfected into STING KO RAW264.7 cells.After treatment with 4-OI,it was verified that 4-OI modified STING cysteine mainly through alkylation.Inhibition of STING dimerization affects STING phosphorylation.3.WT and IRG1 KO mice were intraperitoneally injected with itaconate or 4-OI,and then cecal ligation and perforation were performed to establish an abdominal infection model.The survival rate of mice,the phosphorylation level of STING downstream protein TBK1,the changes of inflammatory factors and the changes of intestinal tissue damage were observed.The cecal ligation and perforation of STING KO mice were performed to construct the model of abdominal infection,and the role of STING in the development of sepsis was verified.Research results:1.Stimulating STING can promote the activation of IRG1itaconate2.4-OI alkylation modifies cysteine 147 of STING and inhibits STING phosphorylation3.Itaconate and its derivative,4-OI,down-regulate inflammation in mouse models of sepsis Research conclusions and significance: we find that itaconate,an endogenous immunomodulator,can significantly inhibit the activation of STING signaling.Moreover,4-octyl itaconate(4-OI),which is a permeable itaconate derivative,can alkylate cysteine sites 65,71,88 and 147 of STING,thereby inhibiting its phosphorylation.Furthermore,itaconate and 4-OI inhibit the production of inflammatory factors in sepsis models.Our results have broadened the knowledge on the role of the IRG1-itaconate axis in immunomodulation and highlighted itaconate and its derivatives as potential therapeutic agents in sepsis.Figure 6 Table 1 Reference 34...