Comparative evaluation of metabolic, electrophilic, and immunologic effects of itaconate and its ester derivatives on macrophage activation

Itaconate is a metabolite naturally produced in large amounts in macrophages after exposure to immune stimulation. Following the discovery of itaconate's immunoregulatory properties, several ester derivatives of this dicarboxylic acid were designed to further examine its role. We evaluated the metabolic, electrophilic, and immunologic profiles of macrophages treated with unmodified itaconate and a panel of itaconate derivatives. We found that only unmodified itaconate strongly enhances LPS-induced IFNb secretion after 12h pre-treatment. Thus, we compared the transcriptional profile of wild type and Irg1-/- cells, which are unable to produce itaconate, after 24h LPS stimulation. We find that cells lacking itaconate have impaired downstream type I interferon signaling, and that addition of itaconate Irg1-/- BMDMs restores this signaling back to levels comparable to wild type. This data highlights the role of itaconate as an immunoregulatory metabolite, and highlights the importance of using unmodified itaconate or genetic knockout models in future mechanistic studies. Overall design: BMDMs were prepared from 6 to 16-week-old mice as described and seeded at 100,000 cells 100 mL in 96 well tissue-culture plates in RMPI-1640 medium supplemented with 10% FBS, 2 mM L-glutamine, and 100 U/mL penicillin-streptomycin. Cells were stimulated with LPS (100 ng/mL; Sigma, E. coli 0111:B4) for 24 hours. For the Irg1 KO + ITA condition, 1 mM itaconate was added to the cells 4 hours after addition of LPS to mimic the natural production of itaconate in wild type cells, which increases from 4 – 24 hours after LPS stimulation. After stimulation, cells were washed with PBS, and directly resuspended in Lysis/Binding Buffer (Dynabeads mRNA DIRECT Kit, Invitrogen).