72: Critical role for post-transcriptional regulation of the gene encoding the key inflammasome protein NLRP3

Abstract

The NLRP3 inflammasome regulates the maturation of the pro-inflammatory cytokines IL-1 β and IL-18, which are important mediators in inflammatory disorders such as atherosclerosis, Type 2 diabetes and Alzheimer’s disease. Given its role as an inflammatory gatekeeper, NLRP3 expression is tightly controlled. It is transcriptionally regulated by NF- κ B as well as post-translationally by S-nitrosylation and ubiquitination. We decided to test whether NLRP3 is also regulated at the post-transcriptional level. We found that the NLRP3 3’ untranslated region (UTR) contains an evolutionarily conserved target site for miRNA-223 and we confirmed miR-223 as a direct negative regulator of NLRP3 using a luciferase reporter assay. Furthermore, overexpressing the miRNA reduces NLRP3 levels and IL-1 β production following activation with NLRP3 agonists. Since miR-223 is differentially expressed during myeloid differentiation, it could alter the threshold for inflammasome activation in different cell types. We have now generated a transgenic mouse deficient in the miR-223 target site in the NLRP3 3’UTR, which will allow us for the first time to study the importance of miRNA regulation of NLRP3 in vivo and in the context of inflammatory disease models. Interestingly, the human NLRP3 3’UTR can be alternatively polyadenlyated, giving rise to a truncated form of the transcript that lacks regulatory elements. While mRNA stability was not affected, a luciferase reporter assay showed that LPS stimulation could enhance expression of both 3’UTR isoforms. Using an RNA pulldown approach we identified potential RNA binding proteins that could mediate this effect. Ultimately, understanding the post-transcriptional control of NLRP3 expression will help to explain cell type-specific expression of NLRP3 and could potentially provide new opportunities to interfere with inflammasome activation in diseases such as Type 2 diabetes.