Fig. 4. IRF5-KO alters TCA cycle metabolite concentrations and mitochondrial structural components in macrophages in response to lipotoxicity.

Bone-marrow-derived macrophages (BMDMs) from WT and IRF5-KO mice were treated with either bacterial lipopolysaccharides (LPS) or palmitate (Palm) for 2 or 24 h. Targeted metabolomics analyses were carried out to quantify intracellular tricarboxylic acid (TCA) cycle metabolites (n = 5–6 per condition) and electron microscopy was carried out to evaluate mitochondrial structural characteristics (n = 3 per condition). a Principal component analysis (PCA) on TCA cycle metabolites in all conditions. Palm 2 h condition separated (right). b PCA on TCA cycle metabolites in WT and IRF5-KO BMDMs stimulated with Palm for 2 h. c Variable weighting from PCA, percent variance contribution to principal component (PC)1, upon 2 h of Palm treatment. Lactate (Lac) intracellular concentration in WT and IRF5-KO BMDMs treated with Palm for 2 h (n = 6 per condition, **p = 0.003, two-tailed unpaired t-test). Aco aconitate, Gln glutamine, Gly glycerate, Asp aspartate, PEP phosphoenol pyruvate, Suc succinate, 3PG 3-phospho glycerate, AKG a-ketoglutarate, AHG a-hydroxyglutarate, Mal malate, Pyr pyruvate, Fum fumarate, Ita itaconate, Glt glutamate. d Schematic representation of metabolites with increased (red) or decreased (blue) abundance in IRF5-KO relative to WT BMDMs following treatment with Palm. Cit citrate, Iso isocitrate. e Extracellular acidification rate (ECAR) from extracellular flux analysis under Basal (Bas) and glucose-stimulated (Glu) conditions, of WT and IRF5-KO BMDMs treated with Palm for 2 h (n = 5 per condition, two-tailed unpaired t-tests, **p = 0.006, *p = 0.01). f Oxygen consumption rate (OCR) from extracellular flux analysis, with oligomycin (Oli), carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) and Rotenone/Antimycin A (Rot/AA) administration, performed on WT and IRF5-KO BMDMs, treated with Palm for 2 h (n = 5 per condition). g Electron micrograph and magnified inlet of BMDMs from WT and IRF5-KO mice after 2 h Palm treatment. Mitochondria are marked by ‘m’. h Mitochondrial cristae (white arrows) in electron micrograph and length and number of cristae of BMDMs from IRF5-KO and WT mice following Palm treatment for 2 h (*p = 0.01, *p = 0.04 unpaired t-test). Cell were derived from n = 3 independent animals per genotype and individual mitochondria were analysed in each sample (n = 26, 23 and 20 for WT and n = 11, 16 and 16 for IRF5-KO). Data presented as mean ± SEM. Source Data file provided.