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Although the immunomodulatory and cytoprotective properties of itaconate have been studied extensively, it is not known whether its naturally occurring isomers mesaconate and citraconate have similar properties. Here, we show that itaconate is partially converted to mesaconate intracellularly and that mesaconate accumulation in macrophage activation depends on prior itaconate synthesis. When added to human cells in supraphysiological concentrations, all three isomers reduce lactate levels, whereas itaconate is the strongest succinate dehydrogenase (SDH) inhibitor. In cells infected with influenza A virus (IAV), all three isomers profoundly alter amino acid metabolism, modulate cytokine/chemokine release and reduce interferon signalling, oxidative stress and the release of viral particles. Of the three isomers, citraconate is the strongest electrophile and nuclear factor-erythroid 2-related factor 2 (NRF2) agonist. Only citraconate inhibits catalysis of itaconate by cis-aconitate decarboxylase (ACOD1), probably by competitive binding to the substrate-binding site. These results reveal mesaconate and citraconate as immunomodulatory, anti-oxidative and antiviral compounds, and citraconate as the first naturally occurring ACOD1 inhibitor.

Drug induced steatosis (DIS) is characterised by excess triglyceride accumulation in the form of lipid droplets (LD) in liver cells. To explore mechanisms underlying DIS we interrogated the publically available microarray data from the Japanese Toxicogenomics Project (TGP) to study comprehensively whole genome gene expression changes in the liver of treated rats. For this purpose a total of 17 and 12 drugs which are diverse in molecular structure and mode of action were considered based on their ability to cause either steatosis or phospholipidosis, respectively, while 7 drugs served as negative controls. In our efforts we focused on 200 genes which are considered to be mechanistically relevant in the process of lipid droplet biogenesis in hepatocytes as recently published (Sahini and Borlak, 2014). Based on mechanistic considerations we identified 19 genes which displayed dose dependent responses while 10 genes showed time dependency. Importantly, the present study defined 9 genes (ANGPTL4, FABP7, FADS1, FGF21, GOT1, LDLR, GK, STAT3, and PKLR) as signature genes to predict DIS. Moreover, cross tabulation revealed 9 genes to be regulated ≥10 times amongst the various conditions and included genes linked to glucose metabolism, lipid transport and lipogenesis as well as signalling events. Additionally, a comparison between drugs causing phospholipidosis and/or steatosis revealed 26 genes to be regulated in common including 4 signature genes to predict DIS (PKLR, GK, FABP7 and FADS1). Furthermore, a comparison between in vivo single dose (3, 6, 9 and 24 h) and findings from rat hepatocyte studies (2 h, 8 h, 24 h) identified 10 genes which are regulated in common and contained 2 DIS signature genes (FABP7, FGF21). Altogether, our studies provide comprehensive information on mechanistically linked gene expression changes of a range of drugs causing steatosis and phospholipidosis and encourage the screening of DIS signature genes at the preclinical stage.

Excessive inflammation is a major cause of morbidity and mortality in many viral infections including influenza. Therefore, there is a need for therapeutic interventions that dampen and redirect inflammatory responses and, ideally, exert antiviral effects. Itaconate is an immunomodulatory metabolite which also reprograms cell metabolism and inflammatory responses when applied exogenously. We evaluated effects of endogenous itaconate and exogenous application of itaconate and its variants dimethyl- and 4-octyl-itaconate (DI, 4OI) on host responses to influenza A virus (IAV). Infection induced expression of ACOD1, the enzyme catalyzing itaconate synthesis, in monocytes and macrophages, which correlated with viral replication and was abrogated by DI and 4OI treatment. In IAV-infected mice, pulmonary inflammation and weight loss were greater in Acod1-/- than in wild-type mice, and DI treatment reduced pulmonary inflammation and mortality. The compounds reversed infection-triggered interferon responses and modulated inflammation in human cells supporting non-productive and productive infection, in peripheral blood mononuclear cells, and in human lung tissue. Itaconates reduced ROS levels and STAT1 phosphorylation, whereas AKT phosphorylation was reduced by 4OI and DI but increased by itaconate. Single-cell RNA sequencing identified monocytes as the main target of infection and the exclusive source of ACOD1 mRNA in peripheral blood. DI treatment silenced IFN-responses predominantly in monocytes, but also in lymphocytes and natural killer cells. Ectopic synthesis of itaconate in A549 cells, which do not physiologically express ACOD1, reduced infection-driven inflammation, and DI reduced IAV- and IFNγ-induced CXCL10 expression in murine macrophages independent of the presence of endogenous ACOD1. The compounds differed greatly in their effects on cellular gene homeostasis and released cytokines/chemokines, but all three markedly reduced release of the pro-inflammatory chemokines CXCL10 (IP-10) and CCL2 (MCP-1). Viral replication did not increase under treatment despite the dramatically repressed IFN responses. In fact, 4OI strongly inhibited viral transcription in peripheral blood mononuclear cells, and the compounds reduced viral titers (4OI>Ita>DI) in A549 cells whereas viral transcription was unaffected. Taken together, these results reveal itaconates as immunomodulatory and antiviral interventions for influenza virus infection.

BackgroundNon-alcoholic fatty liver disease (NAFLD) is a major health burden in need for new medication. To identify potential drug targets a genomic study was performed in lipid-laden primary human hepatocyte (PHH) and human hepatoma cell cultures.MethodsPHH, HuH7 and HepG2 hepatoma cell cultures were treated with lipids and/or TNFα. Intracellular lipid load was quantified with the ORO assay. The Affymetrix HG-U133+ array system was employed to perform transcriptome analysis. The lipid droplet (LD) growth and fusion was determined by fluorescence microscopy. LD associated proteins were imaged by confocal immunofluorescence microscopy and confirmed by Western immunoblotting. Bioinformatics defined perturbed metabolic pathways.ResultsWhole genome expression profiling identified 227, 1031 and 571 significant regulated genes. Likewise, the combined lipid and TNFα treatment of PHH, HuH7 and HepG2 cell cultures revealed 154, 1238 and 278 differentially expressed genes. Although genomic responses differed among in-vitro systems, commonalities were ascertained by filtering the data for LD associated gene regulations. Among others the LD-growth and fusion associated cell death inducing DFFA like effector C (CIDEC), perilipins (PLIN2, PLIN3), the synaptosome-associated-protein 23 and the vesicle associated membrane protein 3 were strongly up-regulated. Likewise, the PPAR targets pyruvate-dehydrogenase-kinase-4 and angiopoietin-like-4 were up-regulated as was hypoxia-inducible lipid droplet-associated (HILPDA), flotilin and FGF21. Their inhibition ameliorates triglyceride and cholesterol accumulation. TNFα treatment elicited strong induction of the chemokine CXCL8, the kinases MAP3K8, MAP4K4 and negative regulators of cytokine signaling, i.e. SOCS2&SOCS3. Live cell imaging of DsRED calreticulin plasmid transfected HuH7 cells permitted an assessment of LD growth and fusion and confocal immunofluorescence microscopy evidenced induced LD-associated PLIN2, CIDEC, HIF1α, HILPDA, JAK1, PDK4 and ROCK2 expression. Notwithstanding, CPT1A protein was repressed to protect mitochondria from lipid overload. Pharmacological inhibition of the GTPase-dynamin and the fatty acid transporter-2 reduced lipid uptake by 28.5 and 35%, respectively. Finally, a comparisons of in-vitro/NAFLD patient biopsy findings confirmed common gene regulations thus demonstrating clinical relevance.ConclusionThe genomics of fat-laden hepatocytes revealed LD-associated gene regulations and perturbed metabolic pathways. Immunofluorescence microscopy confirmed expression of coded proteins to provide a rationale for therapeutic intervention strategies. Collectively, the in-vitro system permits testing of drug candidates.Electronic supplementary materialThe online version of this article (10.1186/s12920-018-0438-7) contains supplementary material, which is available to authorized users.

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Itaconate is derived from the tricarboxylic acid (TCA) cycle intermediate cis-aconitate and links innate immunity and metabolism. Its synthesis is altered in inflammation-related disorders and it therefore has potential as clinical biomarker. Mesaconate and citraconate are naturally occurring isomers of itaconate that have been linked to metabolic disorders, but their functional relationships with itaconate are unknown. We aimed to establish a sensitive high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay for the quantification of itaconate, mesaconate, citraconate, the pro-drug 4-octyl-itaconate, and selected TCA intermediates. The assay was validated for itaconate, mesaconate, and citraconate for intra- and interday precision and accuracy, extended stability, recovery, freeze/thaw cycles, and carry-over. The lower limit of quantification was 0.098 µM for itaconate and mesaconate and 0.049 µM for citraconate in 50 µL samples. In spike-in experiments, itaconate remained stable in human plasma and whole blood for 24 and 8 h, respectively, whereas spiked-in citraconate and mesaconate concentrations changed during incubation. The type of anticoagulant in blood collection tubes affected measured levels of selected TCA intermediates. Human plasma may contain citraconate (0.4–0.6 µM, depending on the donor), but not itaconate or mesaconate, and lipopolysaccharide stimulation of whole blood induced only itaconate. Concentrations of the three isomers differed greatly among mouse organs: Itaconate and citraconate were most abundant in lymph nodes, mesaconate in kidneys, and only citraconate occurred in brain. This assay should prove useful to quantify itaconate isomers in biomarker and pharmacokinetic studies, while providing internal controls for their effects on metabolism by allowing quantification of TCA intermediates.

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