Nuclear Localization of IRG1 Modulates COX2 Expression and Prostaglandin Biosynthesis in Primary Human Macrophages 2308479

Abstract

Abstract Introduction Immunometabolism within macrophages represents a dynamic process modulated by environmental factors and pathological conditions. Immune Responsive Gene 1 (IRG1) catalyzes the conversion of cis-aconitate, an intermediate in the tricarboxylic acid (TCA) cycle, to itaconate, and is robustly upregulated in macrophages following toll-like receptor engagement or type II interferon stimulation. The upregulation of IRG1 results in reduced macrophage cytokine production and influences polarization. To date, IRG1 localization has primarily been associated with mitochondria. In this study, we present evidence that IRG1 is also present in the nucleus of macrophages, where it associates with several promoters, including one controlling expression of PTGS2 (COX2), the rate-limiting enzyme in prostaglandin synthesis. Methods In these studies, we employed a multi-omic strategy incorporating ChIP-seq, RNA-seq, global proteomics, and metabolomics. To establish the biological significance of this novel IRG1 function, we generated CRISPR knockouts in human monocyte-derived macrophages and utilized a DSS-induced colitis model to assess inflammation in vivo. Results Through a multi-omic approach encompassing ChIP-seq, RNA-seq, global differential proteomics, and metabolomics, we have elucidated a nuclear function for IRG1 in primary human macrophages influencing prostaglandin biosynthesis. Additionally, investigations using a CRISPR-generated catalytically inactive IRG1 point mutant in THP1 cells demonstrated significant binding of IRG1 to the PTGS2 promoter, resulting in increased COX2 transcript and protein expression independent of itaconate production. Conclusion Collectively, these results reveal a novel, nuclear role for IRG1 in macrophages that provides a deeper understanding of how IRG1 may regulate macrophage function in the context of inflammation. Funding Source Eli Lilly and Company Topic Categories Cellular Adhesion, Migration, and Inflammation (CAM)