An emerging concept in immunology is that rewiring of macrophage metabolism is crucial for controlling their activities. Remodelling of the TCA cycle in macrophages leads to the accumulation of metabolites that can promote proinflammatory or anti-inflammatory functions, but the underlying mechanisms are not fully understood. Now, a study in Nature reports that the metabolite itaconate regulates the key anti-inflammatory transcription factor nuclear factor erythroid 2-related factor 2 (NRF2) in macrophages.
“KEAP1 was alkylated on multiple cysteines and a lysine by itaconate”
The authors first confirmed previous observations by showing that lipopolysaccharide (LPS) upregulates itaconate levels in bone marrow-derived macrophages (BMDMs). The increased itaconate levels were accompanied by elevated levels of NRF2, which activates an antioxidant and anti-inflammatory programme in macrophages. Itaconate has been shown to inhibit the proinflammatory mitochondrial enzyme succinate dehydrogenase (SDH), but a potent SDH inhibitor did not activate NRF2, suggesting that itaconate activates NRF2 by a different mechanism. To study this further, the authors synthesized a new cell-permeable derivative of itaconate, 4-octyl itaconate (OI), which was as effective as other compounds in activating NRF2 and, importantly, was efficiently metabolized to itaconate in cells. To identify how NRF2 is stabilized, the authors examined a known NRF2 regulator, kelch-like ECH-associated protein 1 (KEAP1), which binds to cytosolic NRF2 and promotes its degradation by the proteasome. Using mass spectrometry, the authors showed that KEAP1 was alkylated on multiple cysteines and a lysine by itaconate in OI-treated macrophages. Cysteine 151 is uniquely required for inhibiting KEAP1-mediated degradation of NRF2 and the authors found that mutating this residue in KEAP1 prevented the stabilization of NRF2 by OI. This result suggested that itaconate upregulates NRF2 levels via a newly identified post-translational modification, namely dicarboxylation of KEAP1.
The authors confirmed that the activation of NRF2 by OI has anti-inflammatory effects. Treatment of LPS-stimulated BMDMs with OI reduced their production of pro-IL-1β, IL-10, hypoxia-inducible factor 1α (HIF1α), reactive oxygen species and nitric oxide, but had no effect on NF-kB activity or TNF levels. Furthermore, in an LPS-induced mouse model of sepsis, OI prolonged survival, which was associated with decreased levels of IL-1β and TNF, but not of IL-10.
Finally, modelling of gene networks suggested that the LPS-induced increase in itaconate occurs through type I interferon (IFN)-mediated regulation of the enzyme that synthesizes itaconate, immune-responsive gene 1 protein (IRG1). IFNβ treatment of BMDMs increased their levels of itaconate as well as Irg1 mRNA, which required the presence of the type I IFN receptor. Notably, OI inhibited IFN responses, including IFN-stimulated gene (ISG) expression and IFNβ production, in LPS-activated BMDMs. These results suggest the existence of a negative-feedback mechanism: LPS induces itaconate production, partly via the induction of type I IFNs; this results in NRF2 stabilization (via itaconate-mediated cysteine alkylation of KEAP1) that promotes an anti-inflammatory response, which is accompanied by the downregulation of itaconate production through reduced IFN expression. Thus, itaconate and OI might be interesting potential therapeutics for the treatment of inflammatory diseases.
Mills, E. L. et al. Itaconate is an anti-inflammatory metabolite that activates Nrf2 via alkylation of KEAP1. Nature https://doi.org/10.1038/nature25986 (2018)
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Nature Communications (2019)