Comparative evaluation of itaconate and its derivatives reveals divergent inflammasome and type I interferon regulation in macrophages

Abstract

Upon activation, macrophages undergo extensive metabolic rewiring 1 , 2 . Production of itaconate through the inducible enzyme IRG1 is a key hallmark of this process 3 . Itaconate inhibits succinate dehydrogenase (SDH) 4 , 5 , has electrophilic properties 6 , and is associated with a change in cytokine production 4 . Here, we compare the metabolic, electrophilic, and immunologic profiles of macrophages treated with unmodified itaconate and a panel of commonly used itaconate derivatives to examine its role. Using wild type and Irg1 −/− macrophages, we show that neither dimethyl itaconate (DI), 4-octyl itaconate (4OI), nor 4-monoethyl itaconate (4EI) are converted into intracellular itaconate, while exogenous itaconic acid readily enters macrophages. We find that only DI and 4OI induce a strong electrophilic stress response, in contrast to itaconate and 4EI. This correlates with their immunosuppressive phenotype: DI and 4OI inhibit IκBζ and pro-IL-1β induction, as well as IL-6, IL-10, and IFN-β secretion in an Nrf2-independent manner. In contrast, itaconate treatment only suppressed IL-1β secretion but not pro-IL-1β levels, and, surprisingly, strongly enhanced LPS-induced IFN-β secretion. Consistently, Irg1 −/− macrophages produced lower levels of interferon and reduced transcriptional activation of this pathway. Our work establishes itaconate as an immunoregulatory, rather than strictly immunosuppressive metabolite, and highlights the importance of using unmodified itaconate in future studies.

Benchmarking non-targeted metabolomics using yeast derived libraries

“…Phaff 1956 (Komagataella phaffii Kurtzman) 27 Literature search confirmed our findings and additionally expanded the reported compounds [30][31][32][33][34][35][36][37][38][39] in our library which can be found in the Supplementary two participating laboratories or others [30][31][32][33][34][35][36][37][38][39] ). The remaining 25 metabolites were reported elsewhere [30][31][32][33]36,37 ( Supplementary Table S1, column, FinalMetaboliteList, "Additional literature hits"). The human metabolome database (HMDB) 43,44 was interrogated regarding overlaps with the yeast metabolome data base which is based on the Baker's/Brewer's yeast Saccharomyces cerevisiae 45 crosschecking our library for plausibility.…”

“…Over the recent past, yeast-based standards have seen a slow but increasing acceptance in the metabolomics community. Several laboratories resorted to Pichia pastoris ethanolic yeast extracts [30][31][32][33][34][35][36][37][38][39] primarily for 13 C internal standardization. Only a few studies were related to instrument performance tests or method development in accordance to the here proposed application 33,[38][39][40] .…”

Electrophilic Nrf2 activators and itaconate inhibit inflammation at low dose and promote IL-1β production and inflammatory apoptosis at high dose

“…4-OI has been reported to be hydrolyzed to itaconate by cellular esterases in LPS-activated macrophages, thus making it a suitable cell-permeable surrogate of itaconate [ 9 ]. However, a recent study proposed that 4-OI does not generate free itaconate and that any increase is due to endogenous itaconate being generated (for instance through immune‐responsive gene 1 [Irg1] function) [ 49 ]. To test this latter hypothesis in our system, we generated BMDCs from WT and Irg1 -deficient animals, pulsed them with 4-OI followed by stimulation with LPS and ATP ( Fig.…”

“…How exactly 4-OI exerts its anti- and pro-inflammatory functions remains controversial. Indeed, it has been originally shown that 4-OI is hydrolyzed to itaconate by esterases in LPS-activated macrophages [ 9 ], but a recent publication proposed that any increase in itaconate upon 4-OI treatment is due to endogenous itaconate being generated, for instance through Irg1 activity [ 49 ]. Our results with Irg1 -deficient BMDCs excluded the possibility that 4-OI acts by increasing Irg1-dependent production of endogenous itaconate in our system, thus in keeping with the possibility that 4-OI exerts its anti- and pro-inflammatory bioactivities by itself or by a direct conversion into itaconate via cellular esterases.…”

Small molecule resensitizesSalmonellato itaconate and decreases bacterial proliferation in macrophages

“…This concentration is known to recapitulate the effects of endogenous itaconate expression. 23 After a 10-minute challenge of BV2 cells to S. Typhimurium (at a multiplicity of infection or MOI of 30, i.e. 30 Salmonella cells per BV2 cell), compound 1, compound 2, or the vehicle alone, was added to the growth medium and the cells were allowed to proliferate for an additional 24 hours.…”