Fig. 2: Irg1 is upregulated in hepatic macrophages in response to high-fat diet.
a, Transcriptomic analysis of Acod1/Irg1 mRNA expression on cell subsets in mice fed control/standard diet (SD) or WD for the indicated times from a single-cell RNA-seq experiment in Guilliams et al.17 b,c, Whole liver lobes were collected on week 12 following control or WD feeding of the indicated mice. Murine Irg1 gene expression was quantified in each sample using qPCR. For b, n = 20 wild-type mice per group; n = 6 Cre-specific mice per group (****P < 0.0001; **P = 0.002). For c, n = 5 mice per group (**P = 0.003; ***P = 0.0005). d,e, F4/80+ macrophages were isolated by magnetic selection from single-cell suspensions of liver cells. Graphs depict the relative area of itaconate production in positively selected F4/80+ macrophages (***P = 0.0002; ****P < 0.0001; NS, not significant) (d) and the flow-through non-macrophage fraction (n = 5 mice per group) (e). f, Itaconate production was quantified from homogenized livers of 12 week WD-fed mice, normalized for liver tissue weight (n = 5 mice per group; P = 0.008). g, Itaconate production was quantified from mice treated with 50 μg LPS overnight (n = 3 mice per group; P < 0.0001). h, Secreted itaconate in the supernatants of untreated and LPS-treated hepatocytes or RAW 264.7 cells as a positive control (n = 3 per group). A two-sided ANOVA with multiple comparisons was used for statistical analyses (b–g). i,j, Human Irg1 expression (*P = 0.03) (i) and itaconate production (j) (***P = 0.017) in human NASH livers. Human liver samples from 10 non-NASH controls (consisting of hemangioma, focal nodular hyperplasia, hepatic adenoma, hepatocellular carcinoma and benign cases) and 16 NASH cases were analyzed for itaconate expression using mass spectrometry. Results for Irg1 are shown as the relative fold change over non-NASH controls. Results for itaconate were normalized per mg liver tissue. A two-sided Mann–Whitney U-test was used for statistical analysis of human NASH samples (i,j). k, Itaconate levels in the plasma of non-NASH controls and NASH cases. Data are presented as mean ± s.e.m.
