Mice with Nlrp3 mutations were developed independently by investigators in two laboratories. One group introduced a R258W mutation in the third exon of the Nlrp3 gene of C57BL/6 mice 9. This corresponds to the R260W mutation frequently found in humans

with the Muckle–Wells syndrome 7. A second group introduced either an A350V or an L351P mutation in exon BKM120 supplier 3 of Nlrp3 in 129SvJ mice 10. These mutations occur frequently in patients with Muckle–Wells syndrome and familial cold autoinflammatory syndrome, respectively 10. The targeting strategy used to obtain these strains required that the mice co-express Cre-recombinase to delete a neomycin cassette inserted in reverse orientation that when present causes gene silencing. This allowed studies of mice in which the Cre-recombinase was expressed under tissue-specific promoters and thus enabled tissue-specific expression of the mutated gene 10. In studies

to determine if the R258W mice exhibit the basic immunologic abnormality of patients with CAPS, BM-derived macrophages and BM-derived dendritic cells (BMDC) from these mice were stimulated with a TLR ligand (LPS) in the presence and absence of ATP, the latter an essential co-factor in NLRP3 inflammasome activation in WT cells. It was shown that while cells from R258W mice were unable to produce IL-1β and IL-18 in the absence of stimulation, they produced large amounts of these cytokines upon LPS stimulation in the presence or absence of exogenous ATP. These cells therefore differed from WT cells in that the latter only exhibited IL-1β production upon LPS stimulation in the presence of ATP and thus were similar to cells of patients Bcl-2 inhibitor aminophylline with CAPS. Interestingly, both WT and R258W cells produced equivalent amounts of other cytokines upon LPS stimulation. This suggested that the abnormality was limited to the NLRP3 inflammasome and that elevations in non-inflammasome cytokine production occurring during prolonged inflammation was due to secondary stimulation of cells by increased levels of IL-1β

6, 9. In parallel studies of peritoneal macrophages and BMDC from the A350V and L351P knock-in (KI) mice, production of IL-1β in the absence of ATP was also found. In addition, it was shown that BMDC from L351P mice secreted IL-1β when incubated at 32°C, as do CAPS patients with similar mutations. Thus, cold conditions seem to be an inflammasome activator in the presence of this mutation. Finally, cold-challenged dendritic cells from L351P KI mice exhibited spontaneous IL-1β secretion, whereas A350V KI cells were more dependent on LPS priming; this may explain the greater neonatal mortality of the L351P KI mice when compared with A350V KI mice 10. The mechanism of ATP co-activation of the NLRP3 inflammasome was studied in the R258W KI mice. Previous work has shown that this ATP function is an extracellular activity that involves activation of a membrane receptor, P2X7R 11.