Anti-inflammatory; Nrf2; KEAP1

To overcome the limitations of dimethyl itaconate (DMI), researchers synthesized 4-Octyl itaconate (OI) , a cell-permeable itaconate derivative (Extended Data Fig. 3a). Itaconate and OI had similar thiol reactivity that was far lower than that of DMI (Extended Data Fig. 3b, c, f), making it a suitable cell-permeable itaconate surrogate. Furthermore, OI was hydrolysed to itaconate by esterases in mouse myoblast C2C12 cells (Extended Data Fig. 3d) and LPS-activated mouse macrophages (Extended Data Fig. 3e). OI boosted Nrf2 levels (Fig. 1e, compare lane 5 to lane 1) and enhanced LPS-induced Nrf2 stabilization (Fig. 1e, compare lane 6 to lane 2), and increased the expression of downstream target genes9, including the anti-inflammatory protein HMOX110 (Fig. 1f, g). We used a quantitative NAD(P)H:quinone oxidoreductase-1 (NQO1) inducer bioassay11,12, to assess the potency of Nrf2 activation by the CD value (concentration required to double the specific enzyme activity) for NQO1, the prototypical Nrf2 target gene. OI (CD value of 2 μM), was more potent than the clinically used Nrf2 activator DMF (CD value of 6.5 μM) (Fig. 1h, Extended Data Fig. 3f). OI stimulated synthesis of the key anti-oxidant GSH (Extended Data Fig. 3g–i). OI also boosted canonical activation of Nrf2 by the pro-oxidant hydrogen peroxide (H2O2) (Extended Data Fig. 3j, k). Importantly, the related octyl esters 4-octyl 2-methylsuccinate and octyl succinate, which are not Michael acceptors, had no effect on Nrf2 activity, confirming the requirement for the itaconate moiety (Extended Data Fig. 3l). Dimethyl malonate, a potent SDH inhibitor4, did not activate Nrf2 (Extended Data Fig. 3m), confirming that Nrf2 activation by OI is independent of SDH inhibition[1].

4-Octyl Itaconate (OI) is a derivative of itaconate that is permeable to cells. 4-Octyl itaconate (OI) inhibits cytokine production and stops lipopolysaccharide-induced mortality in vivo [1].

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4-Octyl itaconate (OI) also counteracted the pro-inflammatory response to LPS in vivo. OI, which activated Nrf2 (Extended Data Fig. 7k), prolonged survival, decreased clinical score and improved body temperature regulation, and decreased IL-1β and TNF levels but not IL-10 in an LPS model of sepsis [1].

KEAP1 cysteine target validation [1]
COS1 cells (2.5 × 105 per well) in 6-well plates were co-transfected with 0.8 μg of Nrf2-V5 and 1.6 μg of wild-type or Cys151S mutant KEAP114, or 1.6 μg of pcDNA. Cells were grown for 21 h then treated with 20 or 100 μM 4-Octyl itaconate (OI) , 5 μM sulforaphane or 0.1% acetonitrile (vehicle) for 3 h. Cell were washed in PBS and lysed in 200 μl of SDS-lysis buffer (50 mM Tris-HCl, pH 6.8, 2% (w/v) sodium dodecyl sulfate (SDS) and 10% (v/v) glycerol). Lysates were sonicated (20 s at 30% amplitude using Vibra-Cell ultrasonic processor) and boiled (3 min), and dithiothreitol (DTT) and Bromophenol blue were added up to 0.1 M and 0.02% (w/v) final concentrations, respectively. Proteins (10 μg) were resolved on a gradient (4–12%) NuPAGE SDS gel, transferred onto nitrocellulose membranes, and immunoblotted with anti-KEAP1, anti-Nrf2 (rabbit monoclonal), and anti-β-actin (mouse monoclonal) antibodies. Horseradish peroxidase (HRP)- or IRDye-labelled secondary antibodies were used interchangeably, followed by either ECL detection or scanning using Odyssey imager (Li-COR).

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GSH/GSSG measurements [1]
BMDMs were plated at 0.1 × 106 cells per ml in opaque 96-well plates. Cells were pre-treated with 4-Octyl itaconate (OI) (125 μM) for 2 h and then stimulated with hydrogen peroxide (100 μM) for 24 h. After 24 h, cell media was removed and the reduced glutathione to oxidized glutathione (GSH/GSSG) ratio was quantified using MyBio GSH/GSSG-Glo Assay (V6611) as per manufacturer’s instructions. Luminescence was quantified using a FLUOstar Optima plate reader.

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Analysis of KEAP1 modification by 4-Octyl itaconate (OI) [1]
Cell lines have not been tested for mycoplasma contamination. Twenty-four hours after transfection, cells were treated with 4-Octyl itaconate (OI) (500 μM) or vehicle control (PBS) for 4 h. Tagged KEAP1 was immunoprecipitated using an anti-Flag antibody and protein A/G beads. After immunoprecipitation, bound KEAP1 was eluted off the beads using Flag peptide (500 μl; 200 μg ml−1) diluted in 1× TBS pH 7.4. The samples were then concentrated and the Flag peptide was removed using 10K centrifugation filter columns. The concentrated samples were then divided in half for downstream processing. One-half of each sample was diluted 1:2 with 5× SDS sample buffer and separated using SDS–PAGE. Overexpressed KEAP1 was detected using Coomassie blue staining and the corresponding bands were excised from the gel and subjected to in-gel digest as described. In brief, the gel slices were cut into smaller pieces (1–2 mm3) before reduction with DTT (10 mM) and alkylation with iodoacetamide (50 mM). Half of the gel slices from each sample were then subjected to a trypsin (2 μg) digest, the other half were digested with elastase (1 μg) overnight at 37 °C. Similarly, the remaining sample concentrates (in solution) were reduced with DTT and alkylated with iodoacetamide, before precipitation of the protein via the methanol–chloroform extraction method. The protein pellet was re-suspended in urea (6 M), which was then diluted to <1 M urea with ultrapure H2O. The samples were then digested with trypsin (2 μg) overnight at 37 °C. Digested protein samples were analysed in an Orbitrap Fusion Lumos coupled to a UPLC ultimate 3000 RSLCnano System.

Metabolite measurements for absolute succinate and itaconate quantification and metabolite tracing [1]
Cells were treated as desired. For tracing studies, immediately before LPS stimulation, the media was removed and replaced with DMEM media (1 ml) containing U-13C-glucose (4.5 g l−1) or U-13C-glutamine (584 mg ml−1) deplete of 12C-glucose or 12C-glutamine. Samples were extracted in methanol/acetonitrile/water, 50:30:20 (v/v/v) (1 ml per 1 × 106 cells) and agitated for 15 min at 4 °C in a Thermomixer and then incubated at −20 °C for 1 h. Samples were centrifuged at maximum speed for 10 min at 4 °C. The supernatant was transferred into a new tube and centrifuged again at maximum speed for 10 min at 4 °C. The supernatant was transferred autosampler vials. Liquid chromatograph–mass spectrometry (LC–MS) analysis was performed using a Q Exactive mass spectrometer coupled to a Dionex U3000 UHPLC system. For further details, see Supplementary Methods.

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Western blotting [1]
Protein samples from cultured cells were prepared by direct lysis of cells in 5× Laemmli sample buffer, followed by heating at 95 °C for 5 min. For spleen samples, 30 mg of spleen was homogenized in RIPA buffer using the Qiagen TissueLyserII system. The resulting homogenate was centrifuged at 14,000 r.p.m. for 10 min at 4 °C, and supernatants were used for SDS–PAGE. Protein samples were resolved on 8% or 12% SDS–PAGE gels and were then transferred onto polyvinylidene difluoride (PVDF) membrane using either a wet or semi-dry transfer system. Membranes were blocked in 5% (w/v) dried milk in TBS-Tween (TBST) for at least 1 h at room temperature. Membranes were incubated with primary antibody, followed by the appropriate horseradish peroxidase-conjugated secondary antibody. They were developed using LumiGLO enhanced chemiluminescent (ECL) substrate. Bands were visualized using the GelDoc system

Mice were euthanized in a CO2 chamber and death was confirmed by cervical dislocation. Bone marrow cells were extracted from the leg bones and differentiated in DMEM (containing 10% fetal calf serum, 1% penicillin/streptomycin and 20% L929 supernatant) for 6 days, at which time they were counted and replated for experiments. Unless stated, 5 × 106 BMDMs per millilitre were used in in vitro experiments. Unless stated, the LPS concentration used was 100 ng ml−1, the DMI and 4-Octyl itaconate (OI) concentration was 125 μM, and in experiments where pre-treatments occurred before LPS stimulation this was for 3 h.[1]

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Endotoxin-induced model of sepsis [1]
For cytokine measurements, mice were treated intraperitoneally with 4-Octyl itaconate (OI) (50 mg kg−1) in 40% cyclodextrin in PBS or vehicle control for 2 h before stimulation with LPS (Sigma; 2.5 mg kg−1) intraperitoneally for 2 h. Mice were euthanized in a CO2 chamber, blood samples were collected and serum was isolated. Cytokines were measured using R&D ELISA kits according to manufacturer’s protocol. For temperature recording, mice (n = 10 per group) were treated intraperitoneally with 4-Octyl itaconate (OI) (50 mg kg−1) in 40% cyclodextrin in PBS or vehicle control for 2 h before stimulation with LPS (5 mg kg−1) and monitored for temperature at 1, 2, 3, 4, 6, 12, 18 and 24 h after LPS treatment. Temperature was monitored using subcutaneously implanted temperature transponder chips (Bio Medic Data Systems; IPTT 300) which were injected between the shoulder blades 48 h before experiment. At defined times, body temperature was measured by scanning the transponder with a corresponding BMDS Smart Probe. Animals were additionally monitored for clinical signs of endotoxic shock, based on temperature change, body condition, physical condition and unprovoked behaviour, with a combined score of 9 indicating the humane end point for the experiment.

[1]. Itaconate is an anti-inflammatory metabolite that activates Nrf2 via alkylation of KEAP1. Nature. 2018 Apr 5;556(7699):113-117.

The endogenous metabolite itaconate has recently emerged as a regulator of macrophage function, but its precise mechanism of action remains poorly understood. Here we show that itaconate is required for the activation of the anti-inflammatory transcription factor Nrf2 (also known as NFE2L2) by lipopolysaccharide in mouse and human macrophages. We find that itaconate directly modifies proteins via alkylation of cysteine residues. Itaconate alkylates cysteine residues 151, 257, 288, 273 and 297 on the protein KEAP1, enabling Nrf2 to increase the expression of downstream genes with anti-oxidant and anti-inflammatory capacities. The activation of Nrf2 is required for the anti-inflammatory action of itaconate. We describe the use of a new cell-permeable itaconate derivative, 4-octyl itaconate, which is protective against lipopolysaccharide-induced lethality in vivo and decreases cytokine production. We show that type I interferons boost the expression of Irg1 (also known as Acod1) and itaconate production. Furthermore, we find that itaconate production limits the type I interferon response, indicating a negative feedback loop that involves interferons and itaconate. Our findings demonstrate that itaconate is a crucial anti-inflammatory metabolite that acts via Nrf2 to limit inflammation and modulate type I interferons.[1]

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OI induction of HMOX1 was blocked in Nrf2-deficient macrophages (Fig. 3h (compare lanes 2 and 3 to lanes 8 and 9) and Extended Data Fig. 8a, d) or when Nrf2 was silenced (Extended Data Fig. 8a, d (compare lanes 7 and 8 to lanes 11 and 12)). Without Nrf2, the decrease in LPS-induced IL-1β with OI was significantly impaired (Fig. 3h (compare lane 6 to lane 12), Extended Data Fig. 8b–f (compare lanes 6 and 8 to 10 and 12 in c, d)). Furthermore, two Nrf2 activators, diethyl maleate and 15-deoxy-Δ12,14-prostaglandin J2 decreased LPS-induced IL-1β, IL-10, nitric oxide synthase (NOS2) and nitrite (Extended Data Fig. 8g–k). Thus, itaconate activates an anti-inflammatory program through Nrf2.[1]

C₁₃H₂₂O₄

242.31

242.151

C, 64.44; H, 9.15; O, 26.41

3133-16-2

14239884

White to off-white solid powder

1.584

1

4

11

17

258

0

KBASUIDPDITQHT-UHFFFAOYSA-N

InChI=1S/C13H22O4/c1-3-4-5-6-7-8-9-17-12(14)10-11(2)13(15)16/h2-10H2,1H3,(H,15,16)

2-methylidene-4-octoxy-4-oxobutanoic acid

4-Octyl Itaconate; OI; 4 Octyl Itaconate; 4-Octyl Itaconate; 3133-16-2; 2-Methylene-4-(octyloxy)-4-oxobutanoic acid; 2-methylidene-4-(octyloxy)-4-oxobutanoic acid; Itaconic acid 4-octyl ester; 4-OctylItaconate; 2-methylidene-4-octoxy-4-oxobutanoic acid; SCHEMBL3681702; 4-Octyl-Itaconate

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~412.69 mM)
H2O : < 0.1 mg/mL

Solubility in Formulation 1: ≥ 2.5 mg/mL (10.32 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (10.32 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (10.32 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: ≥ 2.5 mg/mL (10.32 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 5: ≥ 2.5 mg/mL (10.32 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 6: 1 mg/mL (4.13 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication (<60°C).

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Solubility in Formulation 7: 5 mg/mL (20.63 mM) in 20% HP-β-CD in Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication (<60°C).
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)