NLRP3 activation induces ASC-dependent programmed necrotic cell death, which leads to neutrophilic inflammation

Abstract

NLR family pyrin domain containing 3 (NLRP3) is a cytoplasmic pattern recognition receptor that regulates innate immune responses by forming a protein complex, the inflammasome. It leads to production of proinflammatory cytokine productions such as interleukin 1β (IL-1β). We and others demonstrated that an induction of activated NLRP3 also induced cell death. However, little is known about the characteristics and mechanisms of the cell death and its involvement in the pathogenesis of inflammatory conditions. In this study, we established cell lines in which NLRP3 was induced by doxycycline using a tetracycline-inducible expression (Tet-on) system. Using this system, the expression of NLRP3 mutants in cryopyrin-associated periodic syndrome (CAPS) patients was sufficient for the induction of necrotic cell death without lipopolysaccharide stimulation or generation of mature IL-1β. We also found that CA074-Me, a cathepsin B inhibitor, blocked cell death before oligomerization of apoptosis-associated speck-like protein containing a CARD (ASC), whereas Z-VAD-fmk, a pan-caspase inhibitor, blocked the cell death after the oligomerization. Silencing of the ASC gene (Pycard) by small hairpin RNA treatment inhibited the NLRP3 mutant-induced cell death, but silencing of the caspase-1 gene (Casp1) did not. Taken together, these results indicated that ASC was indispensable for NLRP3-mediated programmed necrotic cell death, and that this type of cell death was distinct from ‘pyroptosis', which requires caspase-1. Finally, we demonstrated in an in vivo model that the programmed necrotic cell death induced by activated NLRP3 could cause neutrophil infiltration, indicating a possible role of cell death in neutrophil infiltration of skin lesions in CAPS patients.

Nuclear Factor E2-Related Factor-2 Negatively Regulates NLRP3 Inflammasome Activity by Inhibiting Reactive Oxygen Species-Induced NLRP3 Priming

“…However, excessive and chronic activation of the inflammasome results in many human diseases, such as fa-NLRP3 expression is relatively low and the assembly of the inflammasome complex is poorly induced. Many exogenous and endogenous factors can promote NLRP3 expression, including TLR agonists, proinflammatory cytokines, and reactive oxygen species (ROS) (37). It is interesting to note that almost all NLRP3 agonists can trigger ROS generation and that oxidative stress plays a vital role in NLRP3 inflammasome activation.…”

Mycobacterium tuberculosis and the human macrophage : shifting the balance through inflammasome activation

“…Several pathogens including Shigella, Neisseria and Klebsiella have shown to induce pyronecrosis (269)(270)332). Furthermore, activating mutations in NLRP3 associated with CAPS also lead to pyronecrotic cell death (332)(333)(334). Pyronecrosis is less well defined than pyroptosis, since morphological features seem to be limited to increased permeability of the plasma membrane, a feature also present during necrosis and pyroptosis (311).…”

“…Pyronecrosis is less well defined than pyroptosis, since morphological features seem to be limited to increased permeability of the plasma membrane, a feature also present during necrosis and pyroptosis (311). It does not include DNA fragmentation and is independent of caspase-1, while being reduced during silencing or knock-out of NLRP3 and ASC (269)(270)(332)(333)(334). Pyronecrotic cell death can be inhibited by inhibiting cathepsin B, suggesting that NLRP3 activation following lysosomal damage is the NLRP3-activating pathway responsible for inducing pyronecrosis.…”

Investigation of cell death caused by the Nanopatch™ and its role in vaccine delivery as a physical immune enhancer

“…NALP3 activation can occur through several stimuli including danger signals such as ATP released by dead (necrotic) cells; it was also shown to be required for protective immunity against Influenza and other pathogens [536]. Further information the reader is referred to excellent reviews [398,403,551,553,554]. As previously mentioned, NALP3, ASC and caspase-1 were identified as essential factors in the adjuvanting effect that alum exerts [95].…”

“…More specifically, the inflammasome complex is composed of the NALP3 scaffold: a NACHT-, LRR-and NALP family member that acts as a sensor for danger signals and the adaptor protein apoptosis-associated speck-like protein containing a CARD domain (ASC), which allows the recruitment of caspase-1 in the complex [187,401,551]. Several NLRs (NALPs and IPAF) together with ASC form a caspase-1-activating multi-protein complex, the inflammasome, which leads to the activation of caspase-1 that cleaves the pro-inflammatory cytokines pro-interleukin-1β (IL-1β) and pro-IL-18 into their active form and their subsequent secretion by activated macrophages [552].…”