Role of the leucine-rich repeat domain of cryopyrin/NALP3 in monosodium urate crystal–induced inflammation in mice

Authors


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.

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