Immunoresponsive Gene 1 and Itaconate Inhibit Succinate Dehydrogenase to Modulate Intracellular Succinate Levels

Abstract

Metabolic reprogramming is emerging as a hallmark of the innate immune response, and the dynamic control of metabolites such as succinate serves to facilitate the execution of inflammatory responses in macrophages and other immune cells. Immunoresponsive gene 1 (Irg1) expression is induced by inflammatory stimuli, and its enzyme product cis-aconitate decarboxylase catalyzes the production of itaconate from the tricarboxylic acid cycle. Here we identify an immunometabolic regulatory pathway that links Irg1 and itaconate production to the succinate accumulation that occurs in the context of innate immune responses. Itaconate levels and Irg1 expression correlate strongly with succinate during LPS exposure in macrophages and non-immune cells. We demonstrate that itaconate acts as an endogenous succinate dehydrogenase inhibitor to cause succinate accumulation. Loss of itaconate production in activated macrophages from Irg1(-/-) mice decreases the accumulation of succinate in response to LPS exposure. This metabolic network links the innate immune response and tricarboxylic acid metabolism to function of the electron transport chain.

Keywords: immunoresponsive gene 1 (Irg1); inflammation; itaconate; macrophage; metabolic regulation; mitochondrial metabolism; mitochondrial respiratory chain complex; succinate; succinate dehydrogenase (SDH).

FIGURE 1.

Succinate and itaconate accumulate in LPS-activated murine RAW 264. 7 macrophages. a, dynamics of itaconate, succinate, citrate, α-ketoglutarate, fumarate, and malate levels. Cells were exposed to 10 ng·ml⁻¹ LPS, and metabolites were extracted every hour over a 6-h period. Graphs represent the mean ± S.E. of time-dependent, intracellular metabolite concentrations [mm] of two repeated experiments, each with three biological replicates. b, the basal OCR is unchanged in LPS-activated (LPS) macrophages compared with resting macrophages (Ctr). Error bars represent mean ± S.E. of two repeated experiments.

FIGURE 2.

Exogenous itaconate drives succinate accumulation. Intracellular TCA cycle intermediate and itaconate quantification in resting (Ctr, continuous line) and LPS-activated (LPS, dashed line) RAW 264.7 macrophages after 6-h exposure to increasing extracellular itaconate concentrations (0, 5, 10, and 25 mm) (a, malate; b, citrate; c, itaconate; d, α-ketoglutarate; e, succinate; f, fumarate). Cells were exposed to 10 ng·ml⁻¹ LPS for 6 h. Graphs represent the mean ± S.E. of intracellular metabolite concentrations [mm] of two repeated experiments, each with three biological replicates. Pearson correlation coefficient (r) represents correlation between intracellular itaconate and TCA cycle intermediate concentrations. g, medium itaconate levels of resting and LPS-activated RAW 264.7 macrophages (10 ng·ml⁻¹ LPS) at 0 h (black) and after 6 h (gray). Data represent the mean ± S.E. of metabolite levels [mm] of three biological replicates. h, intracellular succinate quantification in resting and LPS-activated BMDMs after 6-h exposure to 0 mm (black) or 25 mm (gray) extracellular itaconate. Cells were exposed to 10 ng·ml⁻¹ LPS for 6 h. Graphs represent the mean ± S.E. of succinate concentration [mm] obtained from two different mice, each with three biological replicates. *, p < 0.05; **, p < 0.01.

FIGURE 3.

Itaconate- and Irg1-induced succinate accumulation is not specific to immune cells. a, intracellular itaconate and succinate levels increase in A549 lung adenocarcinoma cells after exposure to increasing exogenous itaconate concentrations (0, 5, 10, and 25 mm). Data represent the mean ± S.E. of metabolite levels [mm] of two repeated experiments with each three biological replicates. b, itaconate levels in medium after 6 h (gray) are not significantly affected compared with 0 h (black). Data represent the mean ± S.E. of metabolite levels of two repeated experiments with three biological replicates each. c and d, intracellular levels of itaconate (c, black) and succinate (d, gray) increased in Irg1 overexpression A549 cells after transient transfection with murine pCMV6-Irg1 overexpression (pmIrg1) plasmid compared with empty pCMV6-Entry (pCMV6) control plasmid. Error bars represent the intracellular metabolite levels (ion counts) 24 h after transfection of three biological replicates (mean ± S.E.). *, p < 0.05; **, p < 0.01.

FIGURE 4.

Itaconate is not metabolized to succinate in RAW 264. 7 macrophages. a, carbon labeling indicating oxidative (black lines) and reductive (gray lines) glutamine metabolism using [1-¹³C]glutamine. Labeled itaconate (M1) is only synthesized through reductive glutamine metabolism (gray), and if it is metabolized to succinate, then it would result in succinate containing one labeled carbon (M1). b, mass isotopomer distribution of itaconate (black) and succinate (white) of LPS-activated RAW 264.7 macrophages after 24-h exposure to [1-¹³C]glutamine tracer and 6-h exposure to 10 ng·ml⁻¹ LPS. The major fraction of labeled itaconate contains one labeled carbon, whereas no labeling was found on succinate. Error bars represent the mean ± S.E. of mass isotopomer levels of three biological replicates. c, carbon labeling of TCA cycle intermediates using [U-¹³C₆]glucose and [U-¹³C₅]glutamine tracers. If exogenous, unlabeled itaconate is metabolized to succinate, then labeling would decrease but does not here. d, mass isotopomer distribution of succinate in resting and LPS-activated RAW 264.7 macrophages after 6-h (black) and 24-h (gray) exposure to exogenous, unlabeled itaconate remains ∼90%, indicating that itaconate is not metabolized to succinate. Cells were prelabeled with [U-¹³C₆]glucose and [U-¹³C₅]glutamine over a period of three subcultures. Error bars represent the mean ± S.E. of mass isotopomer levels of three biological replicates.

FIGURE 5.

Itaconate inhibits SDH. a and b, itaconate inhibits the OCR in (a) RAW 264. 7 macrophages and (b) BMDMs in a dose-dependent manner. Shown are normalized OCRs of resting permeabilized cells exposed to increasing itaconate concentrations (0, 2.5, 5, 10, and 25 mm) with either 10 mm (continuous line) or 2.5 mm (dashed line) succinate. Data represent the mean ± S.E. of three repeated experiments. c, itaconate inhibits SDH of the respiratory chain. Shown is the normalized maximal uncoupled OCR of permeabilized resting RAW 264.7 macrophages exposed to various substrates in the presence of 0 mm (black) or 10 mm (white) itaconate. Data represent the mean ± S.E. of three repeated experiments normalized to conditions with 0 mm itaconate and 10 mm succinate.

FIGURE 6.

Loss of Irg1 decreases itaconate and succinate levels in Irg1 KO BMDMs. a, Irg1 expression levels in LPS-activated (24 h, 10 ng·ml⁻¹ LPS) BMDMs obtained from Irg1 KO and WT mice. Error bars represent expression levels obtained from two independent mice (mean ± S.E.) relative to L27. b and c, itaconate and succinate levels in BMDMs obtained from Irg1 KO and WT mice. Cells were activated for 6 h with 10 ng·ml⁻¹ LPS. Error bars represent mean ± S.E. of metabolite levels [mm] of six biological replicates obtained from two independent mice.

FIGURE 7.

Mechanism of LPS-induced succinate accumulation. Under inflammatory conditions, such as LPS stimulation, mammalian CAD catalyzes the decarboxylation of the TCA cycle intermediate cis-aconitate to produce itaconate. This metabolite contributes to succinate accumulation in macrophages by acting as an endogenous SDH inhibitor.