IRF3 Interacts With NLRP3 And Negatively Regulates Activation Of The NLRP3 Inflammasome In Macrophages

Subjective:NLRP3 inflammasome is an important part of the innate immune system.It responds to microbial infection and cell damage by mediating caspase-1 activation and the secretion of pro-inflammatory cytokines IL-1β/IL-18.Interferon Regulator 3(IRF3)is a key factor that regulates the production of interferon in antiviral innate immunity.At the same time,IRF3 also plays an important role in inflammation and antibacterial immunity.We aim to investigate how Interferon Regulator 3 regulate the NLRP3 inflammasome activation and to provide new therapeutic targets for diseases caused by abnormal activation of NLRP3 inflammasomes.Methods:Peritoneal macrophages from WT and IRF3-/-mice were stimulated with NLRP3 inflammasome activators(ATP/nigericin)to investigate the effect of IRF3 on the activity of NLRP3 inflammasome.qPCR was used to detect the mRNA expression levels of proinflammatory cytokines IL-1β,IL-18,TNF-α and NLRP3;Western blotting was used to detect the level of NLRP3 inflammasome components NLRP3,ASC,Caspase-1 and downstream effector molecules Cleaved-Caspase-1 and Cleaved-IL-1β.Meanwhile,the oligomerization level of ASC and NLRP3 after inflammasome activation was also detected.In order to clarify the specific mechanism of IRF3 regulating NLRP3 inflammasome,we used endogenous and exogenous CoIP and immunofluorescence co-localization methods to detect the interaction between IRF3 and NLRP3 and the specific domains of the combination of the two.At the same time,a model of DSS-induced ulcerative colitis in mice was established to study whether IRF3 regulate colitis through NLRP3 inflammasome.Evaluate the weight change,disease activity index,colon tissue pathology and score of the modeled mice;flow cytometry to detect the proportion of immune cells such as macrophages,neutrophils;The expression levels of NLRP3 inflammasome-related effectors in colon tissue was detected by qPCR;Western Blot was used to detect the expression of NLRP3 inflammasome components and related active molecules in colon tissue.Results:we show that the IRF3 is a negative regulator of NLRP3 inflammasome activation.IRF3 deficiency results in increased the level of proinflammatory cytokines IL-1β,IL-18 and TNF-a.Moreover,IRF3 deficiency led to increased induction of NLRP3 and pro-IL-1β protein level.Consistent with these results,the caspase-1 activation and IL-1β maturation observed in response to various NLRP3 inflammasome-activating stimuli were significantly increased in IRF3-deficient PMs.IRF3 obstructs the oligomerization of ASC and NLRP3 also.This finding was further supported by immunofluorescence experiments showing that the LPS and nigericin-induced ASC specks were significantly increased in number on IRF3-/-PMs.Notably,co-immunoprecipitation showed that the NATCH of NLRP3 is required for interaction with the IAD of IRF3 in HEK293T cells.DSS-treated IRF3-/-mice exhibited considerably less body weight loss,lower disease activity index,and longer colon length presentation as compared to WT controls.H&E staining showed that colonic tissues from WT mice were more severe than IRF3-/-animals.The DSS-treated IRF3-/-mice also showed a decreased infiltration of proinflammatory neutrophils and macrophages in the MLNs.IRF3/-mice also activate more NLRP3 inflammasomes in colon tissue.