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Abstract

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 (CryoΔLRR Z/ΔLRR Z) were generated using bacterial artificial chromosome–based targeting vectors, which allow for large genomic deletions. Bone marrow–derived macrophages from CryoΔLRR Z/ΔLRR Z mice, Cryo−Z/−Z mice, and congenic wild-type (WT) mice were challenged with endotoxin-free 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 synovial-like subcutaneous air pouches. The in vivo inflammatory responses were examined.

Results

Release of interleukin-1β (IL-1β), but not CXCL1 and tumor necrosis factor α, was impaired in CryoΔLRR Z/ΔLRR 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-1β production were attenuated in Cryo−Z/−Z and CryoΔLRR Z/ΔLRR 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 CryoΔLRR Z/ΔLRR Z and Cryo−Z/−Z mice. Hence, the LRR domain of cryopyrin plays a role in mediating MSU crystal–induced inflammation in this model.