Role of the Leucine-Rich Repeat Domain of Cryopyrin/NALP3 in Monosodium Urate Crystal-Induced Inflammation in Mice

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 genetics-based 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(Delta LRR) (Z)/(Delta LRR) (Z)) were generated using bacterial artificial chromosome-based targeting vectors, which allow for large genomic deletions. Bone marrow-derived macrophages from Cryo(Delta LRR) (Z/Delta 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 beta (IL-1 beta), but not CXCL1 and tumor necrosis factor alpha, was impaired in Cryo(Delta LRR Z/Delta 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 beta production were attenuated in Cryo(-Z/-Z) and Cryo(Delta LRR Z/Delta 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(Delta LRR Z/Delta 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.