Itaconate inhibits TET DNA dioxygenases to dampen inflammatory responses

Abstract

The immune-response gene 1 (IRG1) plays a key role in anti-pathogen defense, as deletion of Irg1 in mice causes severe defects in response to bacterial and viral infection, and decreased survival 1, 2 . IRG1 transcription is rapidly induced by pathogen infection and in ammatory conditions primarily in cells of myeloid lineage 3 . IRG1 encodes a mitochondrial metabolic enzyme, aconitate decarboxylase 1 (ACOD1), that catalyzes the decarboxylation of cis-aconitate to produce the anti-in ammatory metabolite itaconic acid (ITA) 4 . Several molecular processes are affected by ITA, including succinate dehydrogenase (SDH) inhibition 5 , resulting in succinate accumulation and metabolic reprogramming 6,7 , and alkylation of protein cysteine residues, inducing the electrophilic stress response mediated by NRF2 and IκBζ 8, 9 and impairing aerobic glycolysis 10 . However, the mechanisms by which ITA exerts its profound antiin ammatory effect still remains to be fully elucidated. Here, we show that ITA is a potent inhibitor of the TET family DNA dioxygenases, which catalyze the conversion of 5-methylcytosine (5mC) to 5hydroxymethylcytosine (5hmC) during the process of active DNA demethylation. ITA binds to the same site of α-ketoglutarate (α-KG) in TET2, inhibiting its catalytic activity. Lipopolysaccharides (LPS) treatment, which induces Irg1 expression and ITA accumulation, inhibits Tet activity in macrophages. Transcriptome analysis reveals TET2 is a major target of ITA in suppressing LPS-induced genes, including those regulated by NF-κB and STAT signaling pathways. In vivo, ITA decreases 5hmC, reduces LPS-induced acute pulmonary edema and lung and liver injury, and protects mice against lethal endotoxaemia in a manner that is dependent on the catalytic activity of Tet2. Our study thus identi es ITA as an immune modulatory metabolite that selectively inhibits TET enzymes to dampen the in ammatory response. MainDeletion of the Irg1 gene or treatment with cell permeable ITA alters the transcriptional signature in response to LPS 2 . We speculated that ITA may impact epigenetics to in uence gene expression, and therefore, we determined the effect of Irg1 expression and ITA accumulation on global histone and DNA de/methylation in transfected HEK293T cells (Extended Data Fig. 1a). We found that ectopic expression of either wild-type or catalytic inactive mutant Irg1 had little effect on mono-, di-, and trimethylation of all ve histone H3 lysine residues (Extended Data Fig. 1b, 1c). In contrast, expression of wild-type Irg1, but not the catalytic inactive mutant, dramatically reduced Tet2-mediated global 5hmC in cells (Fig. 1a and Extended Data Fig. 1d-e). Like α-KG, which is a crucial co-substrate for the activity of TET2, ITA is also a dicarboxylic acid containing a 4-or 5-carboxylate that, in the case of α-KG, forms hydrogen and ionic bonds with H1416, R1896, and S1898 in TET2 11 . Of note, α-KG binds to Fe(II) in a bidentate manner via its C-1 carboxylate and C-2 keto groups, which are lacking in ITA. This raises the possibi...

TET proteins regulate T cell and iNKT cell lineage specification in a TET2 catalytic dependent manner

“…Tet3 flx/flx CD4Cre (mentioned Tet3 KO hereafter) (8) mice and Tet2 -/-Tet3 flx/flx CD4Cre (mentioned Tet2/3 DKO hereafter) (8) mice have been previously described. Tet2 H1795R mice (referred to as Tet2 CD hereafter) were designed and generated by MC, MS, YX and ASB at the UNC Animal Models Core (51). For the immune-phenotyping experiments both male and female mice were used.…”

“…Most of the biological tools (mouse strains) are available at Jackson laboratories and we have provided the relevant stock numbers. The Tet2 CD mice are a new strain (51). Requests regarding the Tet2 CD mice should be addressed to Dr. Baldwin (albert_baldwin@med.unc.edu).…”

“…Notably, TET2 has been suggested to exert additional, noncatalytic roles in regulating gene expression in hematopoietic populations (48,49,73) and potentially in controlling the proliferation of iNKTs (8). To assess if TET2 might play a catalytic independent role in regulating conventional and unconventional T cell lineage specification, we used Tet2 CD mice (Figure 4A) that express full length TET2 protein with point mutations, which compromise iron binding and thus render TET2 catalytically inactive (51). We confirmed the point mutations by Sanger sequencing (Figure 4B).…”

5-methylcytosine turnover: Mechanisms and therapeutic implications in cancer

“…Both fumarate and succinate can decrease 5hmC levels genome-wide through inhibition of TET one and TET3. By working as a co-substrate of α-ketoglutarate, Itaconate (ITA) has also been found to be a potential TET inhibitor targeting the same catalytic site on TET2 protein and leading to TET inhibition in lipopolysaccharide (LPS)-activated macrophages, reducing inflammatory responses ( Chen et al, 2022 ). Dimethyloxalylglycine (DMOG), a small molecule mimicking the α-ketoglutarate cofactor has also been used as a TET3 inhibitor ( Zhang et al, 2019 ).…”

Lack of Major Genome-Wide DNA Methylation Changes in Succinate-Treated Human Epithelial Cells

“…This abnormality is thought to be a tumor-driving event, because it results in major epigenetic perturbations. There are also a few other inhibitors of TET enzymes, which include the synthetic compound C35 [ 16 ] and the metabolite itaconate, a reaction product derived from decarboxylation of cis -aconitate that also has structural similarity to alpha-KG [ 17 ]. One of the end products of the enzymatic TET oxidation reaction is succinate, another TCA intermediate, which is produced from alpha-KG during the TET reaction on methylated DNA.…”