CARD-only proteins (COPs) inhibit uric acid crystal-induced inflammasome activation and ameliorate gout

Abstract The NLRP3 inflammasome mediates the response to monosodium urate (MSU) crystals and is responsible for the debilitating symptoms of gout. ASC is the essential inflammasome adaptor that bridges NLRP3 to caspase-1 and inflammasome assembly is via sequential homotypic PYRIN domain (PYD)-PYD and caspase recruitment domain (CARD)-CARD interactions between NLRP3 and ASC and ASC and caspase-1, respectively. While controlled inflammasome activation is essential for host defense and wound healing, dysregulated and excessive inflammasome responses cause inflammatory diseases. Hence, proper control is key for a balanced inflammasome response. Humans, but not mice, encode a family of 3 small CARD-only proteins (COPs): CARD16, CARD17 and CARD18, but their role in controlling inflammasome responses in vivo is unknown, which is the focus of our study. COP transgenic mice show reduced MSU crystal induced release of IL-1b and consequently ameliorated gout symptoms. Transgenic macrophages and COP expressing THP-1 cells show impaired MSU crystal-mediated NLRP3 inflammasome and caspase-1 activation, cytokine release and pyroptosis, while COP KO results in a hyper response to MSU. Also, COPs show unique binding pattern to the CARD of caspase-1 and ASC in naïve, primed and MSU-activated macrophages and interfere with the essential CARD-CARD-mediated caspase-1 self interaction and the ASC-caspase-1 interaction by a competitive binding mechanism. We demonstrate that COPs are functional in mice and inhibit NLRP3 inflammasome assembly by a competitive binding mechanism that prevents cytokine release and pyroptotis and ameliorates inflammatory disease. Altogether, our findings illustrate that the COPs have a role in maintaining homeostasis. National Institutes of Health. (AI099009 and AR064349 to C.S, AI134030, AI140702 and AI120625 to C.S. and A.D.)