Objective. The mechanism by which monosodium urate monohydrate (MSU) crystals intracellularly activate the cryopyrin inflammasome is unknown. The aim of this study was to use a mouse molecular geneticsbased approach to test whether the leucine-rich repeat (LRR) domain of cryopyrin is required for MSU crystal-induced inflammation.Methods. Cryopyrin-knockout lacZ (Cryo ؊Z/؊Z ) mice and mice with the cryopyrin LRR domain deleted and fused to the lacZ reporter (Cryo ⌬LRR Z/⌬LRR Z ) were generated using bacterial artificial chromosome-based targeting vectors, which allow for large genomic deletions. Bone marrow-derived macrophages from Cryo ⌬LRR Z/⌬LRR Z mice, Cryo ؊Z/؊Z mice, and congenic wild-type (WT) mice were challenged with endotoxinfree MSU crystals under serum-free conditions. Phagocytosis and cytokine expression were assessed by flow cytometry and enzyme-linked immunosorbent assay. MSU crystals also were injected into mouse synoviallike subcutaneous air pouches. The in vivo inflammatory responses were examined. Results. Release of interleukin-1␤ (IL-1␤), but not CXCL1 and tumor necrosis factor ␣, was impaired in Cryo⌬LRR Z/⌬LRR Z and Cryo ؊Z/؊Z mouse bone marrow-derived macrophages compared with WT mouse bone marrow-derived macrophages in response to not only MSU crystals but also other known stimuli that activate the cryopyrin inflammasome. In addition, a comparable percentage of MSU crystals taken up by each type of bone marrow-derived macrophage was observed. Moreover, total leukocyte infiltration in the air pouch and IL-1␤ production were attenuated in Cryo ؊Z/؊Z and Cryo ⌬LRR Z/⌬LRR Z mice at 6 hours postinjection of MSU crystals compared with WT mice. Conclusion. MSU crystal-induced inflammatory responses were comparably attenuated both in vitro and in vivo in Cryo⌬LRR Z/⌬LRR Z and Cryo ؊Z/؊Z mice. Hence, the LRR domain of cryopyrin plays a role in mediating MSU crystal-induced inflammation in this model.

Mutations in NLRP3 (CIAS1) are identified in a continuum of related inflammatory disorders, known as cryopyrinopathies since NLRP3 codes for the protein cryopyrin. Approximately 40% of patients with classic presentation lack mutations in the coding region of NLRP3 suggesting heterogeneity or epigenetic factors. Cryopyrin is a key regulator of proinflammatory cytokine release. Therefore, variations in the NLRP3 promoter sequence may have effects on disease state in patients with cryopyrinopathies and other inflammatory diseases. In this report, we confirmed three 5′-untranslated region splice forms with two separate transcriptional start sites, and identified potential promoter regions and six new DNA promoter variants. One variant is unique to a mutation negative cryopyrinopathy patient and increases in vitro gene expression. Additional studies can now be performed to further characterize the NLRP3 promoter and sequence variants, which will lead to better understanding of the regulation of NLRP3 expression and its role in disease.