Molecular mechanism of programmed necrosis induced by NLRP3-inflammasome

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Mutant-NLRP3 expression by Tet-on system induced necrotic cell death, while WT did not. We showed that CA074-Me(casepsin B inhibitor)and Z-VAD-fmk(pan-caspase inhibitor)blocked the mutant-NLRP3 dependent cell death before and after ASC oligomerization, respectively. Knocking down of ASC and caspase-1 by sh-RNA revealed that ASC was required in NLRP3-induced necrotic cell death, whereas caspase-1 was not. We judged the manner of cell death induced by NLRP3-PYD oligomerization as necrosis from several observations, but when NLRP3-PYD was oligomerized, we observed the activation of caspase-3 and PARP, which is observed in apoptosis and was not observed in mutant NLRP3 expression by tet-on system. This indicates that this NLRP3-PYD oligomerization model cannot be a good model of NLRP3 activation. We performed neutrophil infiltration assays using an air-pouch model. This assay showed that NLRP3-mediated necrotic cell death resulted in a neutrophilic inflammatory response without IL-1β.