| ObjectiveNLRP3 inflammasome is a multi-protein complex, which plays crucial roles in host defense against pathogens. The NLRP3 protein is considered as a rate-limiting factor for the activation of the inflammasome, thus its expression must be tightly controlled to maintain immune homeostasis. However, the molecular mechanisms that modulate NLRP3 expression, especially at the transcriptional level, remains largely unknown. In the present study, we showed that aryl hydrocarbon receptor (AhR) activation inhibits NLRP3 expression, caspase-1 activation and subsequent IL-1β secretion in peritoneal macrophages. Furthermore, we verified that AhR activation suppressed Alum-induced peritonitis in vivo. Therefore, we identified AhR as a negative regulator of NLRP3 inflammasome by inhibiting the transcription of NLRP3. Our data suggested that AhR is a potential target for the intervention of inflammatory diseases with uncontrolled inflammasome activation.Materials and Methods1 AhR influences NLRP3 expression1.1 Mouse peritoneal macrophages were pretreated with dimethylsuphoxide (DMSO) or TCDD for 40 min and then stimulated with LPS for the indicated periods. Protein level expressions of NLRP3, ASC, caspase-1, and IL-1β were detected by Western blot.1.2 Mouse peritoneal macrophages were pretreated with dimethylsuphoxide (DMSO) or TCDD for 40 min, and then stimulated with LPS for the indicated time. Expression of NLRP3, ASC, caspase-1, IL-1β mRNA was detected by RT-PCR.1.3 Mouse peritoneal macrophages were pretreated with increased concentrations of TCDD (2nM, lOnM,20nM,50nM) or FICZ (20nM, 100nM,200nM,500nM) for 40 min, and then stimulated with LPS for 8h. Protein level of NLRP3 was detected by Western blot.1.4 Western blot analysis of protein levels of NLRP3, ASC, Pro-caspase-1, and AhR in mouse peritoneal macrophages transfected with scrambled control siRNA or AhR siRNA for 48h.1.5 Mouse peritoneal macrophages transfected with control siRNA or AhR siRNA were stimulated with LPS for the indicated periods. Expression of NLRP3, ASC, Caspase-1, and IL-1β was detected by Western blot.1.6 Mouse peritoneal macrophages transfected with control siRNA or AhR siRNA were stimulated with LPS for the indicated time.mRNA expression of NLRP3, ASC, Caspase-1, and IL-1β were detected by RT-PCR.2 AhR influences promoter activities of NLRP32.1 RAW264.7 cells transfected with the NLRP3 promoter plasmids -2032 were pretreated with dimethylsuphoxide (DMSO) or TCDD for 40 min and then stimulated with LPS for 6 h. The cell lysates were assayed for luciferase activity.2.2 RAW264.7 cells transfected with the NLRP3 promoter plasmids-2032 were pretreated with DMSO, TCDD, FICZ or L-kynurenine (L-kyn) for 40 min and then stimulated with LPS for 6 h. The cell lysates were assayed for luciferase activity.2.3 RAW-AhR or RAW-Vector cells were transfected with the NLRP3 promoter plasmids -2032 and then stimulated with LPS for 6 h. The cell lysates were assayed for luciferase activity.3 The molecular mechanism of regulation of NLRP3 expression by AhR3.1 RAW264.7 cells transfected with the NLRP3 promoter plasmids -2032,-1434 or -1113 were pretreated with dimethylsuphoxide (DMSO) or TCDD for 40 min and then stimulated with LPS for 6 h.The cell lysates were assayed for luciferase activity.3.2 RAW264.7 cells transfected with the NLRP3 promoter mutants NLRP3 ΔXRE1, NLRP3 ΔXRE2 or NLRP3 ΔXRE3 were pretreated with DMSO or TCDD for 40 min and then stimulated with LPS for 6 h. The cell lysates were assayed for luciferase activity.3.3 RAW264.7 cells transfected with the NLRP3 promoter mutants NLRP3 mt-1, NLRP3mt-2, or NLRP3 mt-3 were pretreated with DMSO or TCDD for 40 min and then stimulated with LPS for 6 h. The cell lysates were assayed for luciferase activity.3.4 Mouse peritoneal macrophages were pretreated with DMSO, TCDD or FICZ for 1h, and the ChIP assay was used to assess the binding of AhR to the XRE in the murine NLRP3 promoter.3.5 Mouse peritoneal macrophages transfected with control siRNA or AhR siRNA were pretreated with DMSO or TCDD for 40 min and then stimulated with LPS for 1h, followed by ChIP assay to assess the binding of AhR to the XRE in the murine NLRP3 promoter.4 AhR influences NLRP3 inflammasome activity4.1 Mouse peritoneal macrophages were pretreated with dimethylsuphoxide (DMSO) or TCDD for 40 min, and then stimulated with LPS for 8 h, followed by ATP, nigericin (Nig) or Alum treatment for the last 30 min. Protein levels of cleaved caspase-1 (p10), cleaved IL-1β(p17), Pro-caspase-1 and Pro-IL-1β in the supernatants (SN) and cell lysates (CL) were detected by Western blot.4.2 Mouse peritoneal macrophages transfected with scrambled control siRNA or AhR siRNA were stimulated with LPS for 8 h and ATP or nigericin (Nig) for the last 30 min. Western blot analysis was performed to detect the protein levels of cleaved caspase-1 (p10), cleaved IL-1b (p17), Pro-caspase-1 and Pro-IL-1β in the supernatants (SN) and cell lysates (CL).4.3 Mouse peritoneal macrophages were pretreated with dimethylsuphoxide (DMSO) or TCDD for 40 min, and then stimulated with LPS for the indicated periods, followed by ATP, nigericin (Nig) or Alum treatment for the last 30 min. The level of IL-1β in the culture supernatants was determined by ELISA.4.4 Mouse peritoneal macrophages were pretreated with increased concentrations of TCDD (2nM,10nM,20nM,50nM) for 40 min, and then stimulated with LPS for 8h, followed by ATP or nigericin (Nig) treatment for the last 30 min. The level of secreted IL-1β in the culture supernatants was determined by ELISA.4.5 Mouse peritoneal macrophages were pretreated with DMSO, TCDD, FICZ or L-kynurenine (L-kyn) for 40 min, and then stimulated with LPS for 8 h, followed by ATP or nigericin (Nig) treatment for the last 30 min. The level of IL-1β in the culture supernatants was determined by ELISA.4.6 RAW-Vector or RAW-AhR cells were stimulated with LPS for 8h and then treated with ATP, nigericin (Nig) or Alum for the last 30 min. The level of IL-1β in the culture supernatants was determined by ELISA.4.7 Mouse peritoneal macrophages transfected with scrambled control siRNA or AhR siRNA were pretreated with DMSO or TCDD for 40 min, and then stimulated with LPS for 8h, followed by ATP, nigericin (Nig) or Alum treatment for the last 30 min. The level of IL-1β in the culture supernatants was determined by ELISA.5 AhR influences Alum-induced peritonitis in vivo5.1 C57BL/6 mice were intraperitoneally injected with dimethylsuphoxide (DMSO), TCDD or FICZ for 40 min, and then were treated with Alum for 12h to induce the peritonitis model. Secretion of IL-1β, TNF-α and IL-6 in the lavage fluid was measured by ELISA.5.2 Mouse peritonitis model was induced as described before. Peritoneal exudate cells (PEC) were collected, lysed and analyzed for their expression of caspase-1 and NLRP3 by Western blot.5.3 Mouse peritonitis model was induced as described before. Absolute number of PECs, neutrophils or Ly6C+ monocytes recruited to the peritoneum were analysed by flow cytometry.Results1 AhR inhibits NLRP3 expression1.1 AhR inhibits NLRP3 expression at both of the protein and mRNA levelMouse peritoneal macrophages were pretreated with dimethylsuphoxide (DMSO) or TCDD for 40 min and then stimulated with LPS for the indicated periods (0,4h,8h) and(0,2h,4h). Western blot and RT-PCR results revealed that NLRP3 expression was significantly decreased, while there is no obvious change of the level of ASC, caspase-1 and IL-1β.1.2 AhR inhibits NLRP3 expression in a dose-dependent mannerMouse peritoneal macrophages were pretreated with increased concentrations of TCDD (2nM, lOnM,20nM,50nM) or FICZ (20nM, 100nM,200nM,500nM) for 40 min, and then stimulated with LPS for 8h.Western blot data revealed that the expression of NLRP3 was significantly decreased when the dose of TCDD or FICZ was increased. These data suggested that NLRP3 was inhibited by AhR in a dose-dependent manner.1.3 Block of AhR increases NLRP3 expression under physiological conditionsMouse peritoneal macrophages were transfected with scrambled control siRNA or AhR siRNA, and further cultured for 48h. Expression of NLRP3, ASC, Pro-caspase-1 and AhR protein were detected by Western blot. Our data showed that when AhR was knockdown by its specific siRNA, the expression of NLRP3 was significantly increased, whereas there was no significant change of the expression levels of ASC, caspase-1 and IL-1β.1.4 Knockdown of AhR significantly increased the NLRP3 expression at both of the protein and mRNA levelMouse peritoneal macrophages transfected with control siRNA or AhR siRNA were stimulated with LPS for the indicated periods. Western blot and RT-PCR data showed that NLRP3 expression was significantly increased, whereas the expression of ASC, caspase-1 and IL-1β did not significantly changed.2 AhR binds to NLRP3 promoter and inhibits NLRP3 transcription2.1 AhR inhitits transcriptional activity of NLRP3 promoterRAW264.7 cells transfected with the NLRP3 promoter plasmids -2032 were pretreated with dimethylsuphoxide (DMSO) or TCDD for 40 min, and then stimulated with LPS for 6h. Luciferase activity assay showed that AhR inhitited transcriptional activity of the NLRP3 promoter.2.2 AhR binds to XRE sepuences in the NLRP3 promoterMouse peritoneal macrophages were pretreated with DMSO, TCDD or FICZ for 1 h, and the ChIP assay was used to assess the binding of AhR to the XRE in the murine NLRP3 promoter. Mouse peritoneal macrophages transfected with control siRNA or AhR siRNA were pretreated with DMSO or TCDD for 40 min, and then stimulated with LPS for 1h. The ChIP assay data showed that AhR bound to the XRE in the murine NLRP3 promoter.3 AhR inhibits NLRP3 inflammasome activity3.1 AhR inhibits cleavage of Pro-caspase-1Mouse peritoneal macrophages were pretreated with dimethylsuphoxide (DMSO) or TCDD for 40 min and then stimulated with LPS for 8 h, followed by ATP, nigericin (Nig) or Alum treatment for the last 30 min.Western blot data showed that AhR significantly inhibited cleavage of Pro-caspase-1.3.2 AhR knockdown promotes cleavage of pro-capase-1 cleavingMouse peritoneal macrophages transfected with scrambled control siRNA or AhR siRNA were stimulated with LPS for 8 h, followed by ATP or nigericin (Nig) treatment for the last 30 min. Western blot data showed that AhR significantly promoted cleavage of Pro-caspase-1.3.3 AhR inhibits secretion of IL-1βMouse peritoneal macrophages were pretreated with dimethylsuphoxide (DMSO) or TCDD for 40 min, and then stimulated with LPS for the indicated periods, followed by ATP, nigericin (Nig) or Alum treatment for the last 30 min. ELISA assay showed that AhR significantly inhibited secretion of IL-1β.3.4 Knockdown of AhR promotes secretion of IL-1βMouse peritoneal macrophages transfected with scrambled control siRNA or AhR siRNA were pretreated with DMSO or TCDD for 40 min, and then stimulated with LPS for 8h, followed by ATP, nigericin (Nig) or Alum treatment for the last 30 min. ELISA data showed that koockdown of AhR significantly promoted IL-1β secretion.4 AhR suppresses Alum-induced peritonitis in vivo4.1 AhR inhibits secretion of IL-1β in the lavage fluidC57BL/6 mice were intraperitoneally injected with dimethylsuphoxide (DMSO), TCDD or FICZ for 40 min, and then intraperitoneally injected with Alum for 12h. ELISA data showed that AhR significantly inhibited secretion of IL-1β in the lavage fluid.4.2 AhR inhibits cleavage of Pro-caspase-1 in the lavage fluidPECs in the lavage fluid of the mice with Alum-induced peritonitis were collected, lysed and analysed for their expression of caspase-1 and NLRP3 by immunoblot. Western blot data showed that AhR significantly inhibited cleavage of Pro-caspase-1.4.3 AhR decreases absolute number of PECs, neutrophils and Ly6C+ monocytes in the lavage fluidAbsolute number of PECs, neutrophils or Ly6C+monocytes recruited to the peritoneum were analysed by fluorescence activated cell sorting using flow cytometry. Our data showed that AhR decreased absolute number of PECs, neutrophils or Ly6C+ monocytes in the lavage fluid of these murine models with peritonitis.Conclusion1. AhR significantly inhibits LPS-induced NLRP3 expression at both of the protein and mRNA level.2. AhR binds to NLRP3 promoter and inhibits NLRP3 transcription.3. AhR inhibits activation of NLRP3 inflammasome.Innovations and significances1. Our work defines AhR as a negative regulator of NLRP3 expression at the transcriptional level. AhR binds to XRE sequences of NLRP3 promoter, and further inhibits NLRP3 transcription and NLRP3 inflammasome activity.2. Our results provide a strategy to downregulate inflammasome activity and suggest that AhR may constitute a potential target for the therapeutic modulation of sterile inflammatory diseases. |
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