Regulation Of NLRP3 Inflammasome Activation By Endoplasmic Reticulum Stress Induced By BCG In Macrophage Cells

Background:Tuberculosis(TB)is one of the most popular bacterial infectious diseases around the world.The main pathogen is Mycobacterium tuberculosis(MTB).Macrophages are not only host cells of Mycobacterium tuberculosis,but also immune cells that phagocytize Mycobacterium tuberculosis in the early stage of infection.Inflammasome is a molecular platform activated by cell infection or stress,which can trigger the maturation of pro-inflammatory cytokines and participate in innate immune defense.Previous studies have shown that endoplasmic reticulum stress(ERS)is involved in the activation ofNLRP3 inflammasome in some diseases.However,it is not clear whether ers is involved in the activation of NLRP3 inflammasome in macrophages infected by Mycobacterium tuberculosis and how to regulate it.Objective:To investigate whether macrophages infected with BCG can activate ERS and NLRP3 inflammasome and whether ERS is involved in the activation of NLRP3 inflammasome.Methods:In this study,THP-1 cells and BMDM cells were used as the research objects,and the attenuated strain BCG of Mycobacterium tuberculosis was used as the experimental material.Western blot,ELISA and immunofluorescence were used to analyze the activation of ERS,IRE1? pathway,NF-?B pathway and NLRP3 inflammasome..Results:1.After BCG infected THP-1 cells and BMDM cells,the expression levels of ers related proteins GRP78,caspase-12 and chop were up-regulated,indicating that BCG infected macrophages can activate ers;NLRP3 inflammasome activation related proteins NLRP3,ASC and IL-18 were up-regulated,indicating that BCG infected macrophages can activate NLRP3 inflammasome.2.Compared with BCG alone group,the expression of NLRP3 inflammasome related proteins was down regulated,the secretion of IL-1 ? was decreased,and the expression of ASC,an important component of NLRP3 inflammasome,was decreased in TUDCA+BCG group,indicating that ers was involved in the activation of NLRP3 inflammasome induced by BCG infected macrophages3.Using tunicamycin as a positive control,the ERS pathway activated by BCG infected macrophages was detected.It was found that all three pathways IRE1 ?,PERK and ATF6 were activated,and IRE1 ? was the most significant upregulated.IRE1 ?is involved in the activation of NLRP3 inflammasome induced by BCG infected macrophages.GSK2850163,an inhibitor of IRE 1?,and IRE1 ? siRNA were used to inhibit the activity of IRE1?,respectively.The effect of GSK2850163 on the activation of NLRP3 inflammasome induced by BCG infected macrophages was detected.It was found that the activation of NLRP3.inflammasome was down regulated.These results indicate that IRE1 ? is involved in the activation of NLRP3 inflammasome induced by BCG infected macrophages.4.BCG infected macrophages can activate NF-?B signal.The activation of NF-?B was downregulated by ERS inhibitor TUDCA,IRE1 ? inhibitor GSK2850163 and IRE1? siRNA,and up-regulated by ers agonist tunicamycin,indicating that the activation of NF-?B signal was positively regulated by IRE1? of ERS.When treated with NF-?B inhibitor,the activation of NLRP3 inflammasome activated by BCG infected macrophages was inhibited,indicating that NF-?B signal is involved in the activation of NLRP3 inflammasome activated by BCG infected macrophages.The activation of NLRP3 inflammasome activated by BCG infected macrophages was not inhibited by IRE 1?nuclease inhibitor treatment,indicating that IRE1? positively regulates the activation of NLRP3 inflammasome activated by BCG infected macrophages through kinase activity.Conclusion:Macrophages infected with BCG can activate ers and NLRP3 inflammasome.ERS is involved in the activation of NLRP3 inflammasome in this process,and IRE1?plays a very significant role in regulating the activation of NLRP3 inflammasome.It is further found that IRE1? can regulate the activation of NLRP3 inflammasome through its kinase activity.