Itaconic acid is a mammalian metabolite induced during macrophage activation

Abstract

Itaconic acid (ITA), or methylenesuccinic acid, is not generally classified as a mammalian metabolite. Using NMR-based metabolomics and (13)C-labeling, we have detected ITA in both macrophage-like VM-M3 and RAW 264.7 tumor cell lines as well as stimulated and unstimulated primary murine macrophages. Macrophage activation by addition of lipopolysaccharide and IFN-γ markedly increased ITA production and secretion. Crude cell extracts synthesize ITA via decarboxylation of cis-aconitate, indicative of a novel mammalian cis-aconitic decarboxylase activity. Our results highlight a previously unidentified biosynthetic pathway related to TCA cycle metabolism in mammalian cells and a novel metabolite that likely plays a role in macrophage-based immune response.

Figure 1

(A) 2D-gHSQC spectrum of methanol extracts of VM-M3 cells (8×10⁶ cells) incubated with 25 mM [¹³C₆]glucose for 12 h. Inset: structure of itaconate. Slices from 2D-gHSQC spectra diagnostic for ITA are shown for extracts of RAW 264.7 cells (10⁷ cells) that were (B) unstimulated or (C) stimulated with 10 ng/mL LPS for 24 h, and extracts from murine macrophages (8×10⁶ cells) that were (D) unstimulated or (E) stimulated by priming with 150 U/mL IFN-γ for 6 h followed by 24 h incubation with 10 ng/mL LPS prior to incubation with 25 mM [¹³C₆]glucose for 12 hours. For spectra (A)–(C), nt=128 and ni=128. For spectra (D) and (E), nt=256 and ni=128.

Figure 2

1D gHSQC spectra of culture media from (A) RAW 264.7 cell cultures stimulated with 10 ng/ml LPS and (B) VM-M3 cell cultures that were incubated with [U-¹³C]glucose for 12 h, then treated to isolate weak acids including ITA (*). 6400 transients were collected for each spectrum.

Figure 3

Representative chromatographic traces for the detection of itaconic acid. (A) Standard of citraconic acid (5 µg/mL), a structural isomer of itaconic acid, has a retention time of 13.2 min and detected mass is 129.0196 (error of 2.3 ppm) compared to the theoretical mass of 129.0193, in negative ion mode. (B) Standard of itaconic acid (5 µg/mL), has a retention time of 12.70 min and detected mass of 129.0195 (1.5 ppm error). (C) Itaconic acid detected in the cellular extract of VM-M3 cell line, with a detected mass of 129.0195 (1.5 ppm error).

Figure 4

ITA labeling schemes and isotopologues distribution in itaconate, aconitate and citrate for the first turn of the TCA cycle in VM-M3 cells incubated for 12 h with [U-¹³C]glutamine or [U-¹³C]glucose. ITA labeling scheme from [U-¹³C]glutamine ((A, )) and [U-¹³C]glucose (B and C) if the cis-aconitic decarboxylase (cADC) pathway is active. [U-¹³C]glucose enters glycolysis and results in ¹³C₃-pyruvate, which can enter the TCA cycle as ¹³C₂-acetyl CoA through pyruvate dehydrogenase (PDH), or as ¹³C₃-oxaloacetate through pyruvate carboxylase (PC). The isotopologuess of ITA synthesized by this pathway should be predominantely ¹³C₁ (PDH flux only, (B, )) or ¹³C₄ (both PC and PDH flux, (C, )). (D) Comparison of isotopologues of itaconic acid, citric acid and aconitate in VM-M3 cells after incubation with [U-¹³C]glucose: isotopologuess resulting from PDH flux , or PC/PDH flux [///].

Figure 5

Cis-aconitate decarboxylase (cADC) activity in RAW 264.7 crude cell lysates. Comparison of ¹H spectra (600 MHz) of stimulated RAW 264.7 crude extracts incubated at 37°C in the presence (A) or absence (B) of 4 mM cis-aconitate for 3 h. The resonance at 5.67 ppm is residual cis-aconitate (c-A).