Macrophage fumarate hydratase restrains mtRNA-mediated interferon production

Metabolic rewiring underlies macrophage effector functions, but the mechanisms involved remain incompletely defined. Here, using unbiased metabolomics and stable isotope-assisted tracing, we show induction of an inflammatory aspartate-argininosuccinate shunt following LPS stimulation. The shunt, supported by increased ASS1 expression, also leads to increased cytosolic fumarate levels and fumarate-mediated protein succination. Pharmacologic inhibition and genetic ablation of the TCA cycle enzyme FH further elevates intracellular fumarate levels, suppresses mitochondrial respiration, and increases mitochondrial membrane potential. RNA sequencing and proteomic analysis demonstrate profound inflammatory effects resulting from FH inhibition. Of note, acute FH inhibition suppresses IL-10 expression leading to increased TNF-α secretion, an effect recapitulated by fumarate esters. Unexpectedly, FH inhibition, but not fumarate esters, also increases IFN-β production through mechanisms that are d..., RNA sequencing 
RNA extraction from cells was carried out using an PurelinkTM RNA kit (Invitrogen) according to the manufacturer’s instructions. BMDMs were treated as required, and following treatments were instantly lysed in 350 μl RNA lysis buffer. Isolated RNA was quantified using a NanoDrop 2000 spectrophotometer, and RNA concentration was normalised to the lowest concentration across all samples with RNAse-free water. If necessary, samples were DNAse-treated after quantification using DNAse I (Thermo Fisher) according to the manufacturer’s instructions. BMDMs (3 independent mice) were treated as indicated and RNA was extracted as detailed. mRNA was extracted from total RNA using poly-T-oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers, followed by the second strand cDNA synthesis. The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection. Quantified lib..., 1. RNA sequencing of lipopolysaccaharide-stimulated (4 h) murine bone marrow-derived macrophages (BMDMs) pre-treated with vehicle (DMSO), 20 micromolar fumarate hydratase inhibitor 1 (FHIN1) or 25 micromolar dimethylfumarate (DMF). Three biological replicates per condition.
1_Fpkm_genename.txt
2. Label-free proteomics of lipopolysaccaharide-stimulated (4 h) murine bone marrow-derived macrophages (BMDMs) pre-treated with vehicle (DMSO), 20 micromolar fumarate hydratase inhibitor 1 (FHIN1) or 25 micromolar dimethylfumarate (DMF) for 3 h. Five biological replicates per condition. 
2_diann.pg_matrix.tsv.txt
3. Metabolomics source data - 11 different metabolomics datasets containing the total ion count (TIC)-normalised signal intensity or fraction labelling for the indicated metabolites. All technical and biological replicates are indicated in the file. All experiment conditions are defined in the manuscript. 
3_Metabolomics_data.xlsx
README file describes the 3 types of data in the dataset ...