Fig 2: IRG1 is not regulated by NO.Mouse IRG1 was purified as detailed in Material & Methods. a, IRG1 purification quality is assessed by Coomassie and Western blotting. b, The activity of purified mouse IRG1 was determined by UV, using HPLC at 210 nm and measured 4 h following addition of cis-aconitate at 37 °C in a 0.2 M sodium phosphate solution. c, HPLC chromatograph example of cis-aconitate and itaconate standard detected by HPLC at 210 nm. d, Corresponding nitrite levels of Fig. 2b measured for each condition involving NO production; data is expressed as mean of n = 4 independent experiments ± SEM. e, Effect of increasing concentration of GSH on itaconate standard set at 50 mM. Data are expressed as mean of n = 4 independent experiments ± SEM; Statistical differences were calculated as a one-way ANOVA with Dunnett’s multiple comparisons test against Itaconate standard with no GSH. f, GSH/GSSH ratio were extracted from metabolomic analysis performed in Gch1 WT and Gch1 KO BMDMs and iNOS KO and WT following 18 h stimulation with LPS/IFNγ1. Data is plotted as mean values of n = 6 mice per group ± SEM and show the ratio of GSH/GSSG in both M(LPS/IFNγ) versus unstimulated macrophages (M(unstimulated)) for both genotypes. A one-way ANOVA with Tukey’s mutliple comparisons test was performed. g, Each cysteine of mCAD was mutated individually and replaced with alanine. A mutant was also created with all cysteines mutated to alanines (ALA5). Mismatches are shown by a small white bar and indicated with arrows. h, Protein levels of IRG1 WT and mutants, iNOS and GAPDH (loading control) following transfection were assessed by Western blotting (n = 3 independent experiments). Statistical significance is indicated as ****p < 0.0001, ***p < 0.001, **p < 0.005, *p < 0.05, ns= non-significant. Source data

Fig 3: iNOS and IRG1 in human macrophages.a, Representative Western blot of 3 showing proteins levels of IRG1 and iNOS measured in HEK cells transfected with human IRG1 (hIRG1) cDNA. b, Table of cycle threshold (Ct) values for each gene of interest (NOS2, ACOD1, and GCH1) in unstimulated (CTRL) or 24 h LPS/IFNγ stimulated of n = 4 human monocyte-derived macrophages (Human MDMs) or n = 3 bone marrow organoids (BM organoids). c, Relative gene expression of ACOD1, NOS2 and GCH1 in human MDMs compared to control. Data is represented in bar charts as mean of n = 4 biological replicates ± SD. Statistical differences were calculated as an unpaired t-test. d, Intracellular levels of itaconate in human MDMs measured by HPLC. Data is represented in bar charts as mean of n = 4 biological replicates ± SD. Statistical differences were calculated as an unpaired t-test. Statistical significance is indicated as ****p < 0.0001, ***p < 0.001, **p < 0.005, *p < 0.05, ns= non-significant. Source data

Fig 4: IRG1 is regulated by iNOS but not by NO.a, Hydrogen peroxide levels were determined in media following HEK cell transfection with IRG1 or iNOS cDNA, in the presence or absence of NOC12. Data are expressed as mean values of n = 4 technical replicates; error bars, s.e.m. Statistical differences were calculated using a one-way ANOVA with Dunnett’s multiple comparisons test. b, Activity of purified mouse IRG1 (detailed in Methods) measured by HPLC following incubation with different NO and nitrite donors: 1 mM nitrite, 1 mM SIN-1, 1 mM NOC12 and NOC18, 1 mM GSNO (+10 mM of GSH or GSSG), 1 mM H2O2, 1 mM DTT, 50 or 500 µM of citrulline and 50 or 500 µM of arginine. Data are expressed as mean values of n = 3–4 independent experiments; error bars, s.e.m. Statistical differences were calculated using a one-way ANOVA with Dunnett’s multiple comparisons test against the CTRL condition. c, HEK cells were transfected with IRG1 WT or its mutants cDNA (C184A, C340A, C387A, C432A, C452A, ALA5) and both intracellular itaconate and nitrite levels were measured in the presence or absence of iNOS cDNA. Data are expressed as mean values of n = 4 independent experiments; error bars, s.e.m. Statistical differences were calculated using a one-way ANOVA with Dunnett’s multiple comparisons test against WT or WT + iNOS, respectively. d, Volcano plot of proteins pulled down by Co-IP and identified by mass spectrometry in M(LPS/IFNγ) showing proteins significantly associated with IRG1 in WT (black dots) or iNOS-KO (blue dots) (n = 4 mice per group) following a two-tailed Student’s t-test set at 0.5. e, IRG1 interactome from WT BMDMs was exposed to STRING analysis to exhibit known or predicted interactions using medium-confidence (0.400) settings. f, iNOS intensity data from mass spectrometry were extracted and expressed as a bar chart of mean values of n = 4 independent experiments; error bars, s.e.m. g, Densitometry of iNOS/IRG1 in IP elute in the iNOS-KO model (n = 3 mice per group). IRG1 was precipitated from the cell pellet. Western blots (Extended Data Fig. 4g) were pre-incubated with red fluorophore secondary anti-IgG rabbit and then re-incubated with rabbit anti-IRG1 (using a green secondary antibody), allowing visual separation of IRG1 (53 kDa) from IgG heavy chains present in the immunoprecipitate. Data are expressed as the mean of n = 3 mice; error bars, s.e.m. Statistical differences were calculated using a one-way ANOVA with Dunnett’s multiple comparisons test against WT. h, Densitometry of iNOS/IRG1 in IP elute in the Gch1-KO model (n = 3 mice). IRG1 was precipitated from the cell pellet. Western blots (Extended Data Fig. 4h) were pre-incubated with red fluorophore secondary anti-IgG rabbit and then re-incubated with rabbit anti-IRG1 (using a green secondary antibody), allowing visual separation of IRG1 (53 kDa) from IgG heavy chains in the immunoprecipitate. Data are expressed as the mean of n = 3 mice; error bars, s.e.m. Statistical differences were calculated using Dunnett’s multiple comparisons test against WT. Statistical significance is indicated as ****P < 0.0001; **P < 0.005; *P < 0.05.Source data

Fig 5: Itaconate accumulation is inhibited by iNOS.a, Itaconate levels measured in cell lysate (pink) or media (purple) from BMDM culture using HPLC at specific time points following LPS/IFNγ stimulation (n = 6 mice per group). Nitrite (orange) measured in media by Griess assay, and superoxide anion (blue) in cell pellets by HPLC from WT (n = 7 mice) and iNOS-KO (n = 4 mice) BMDMs. Data are presented as mean values; error bars, s.d. 2-OH-E+, 2-hydroxyethidium. b, Data at 6 h and 18 h were extracted and plotted as a bar chart. Statistical differences were calculated using a two-way analysis of variance (ANOVA) with Šídák’s multiple comparisons test. ND, not detected. c, Densitometry of IRG1/GAPDH is shown (n = 3 mice). Data are expressed as mean values; error bars, s.e.m. Statistical differences were determined by a two-way ANOVA with Šídák’s multiple comparisons test for each time point; see Extended Data Fig. 1c for a representative western blot. d, HEK cells transfected with iNOS and IRG1 cDNAs and treated with iNOS inhibitors (1400W (10 µM) or aminoguanidine (AG; 1 mM)) or NO donors (NOC18 (50 µM) or SIN-1 (100 µM)). Itaconate was measured in cell pellets and nitrite production in media. Data are represented in bar charts as means of n = 5 independent experiments; error bars, s.e.m. Statistical differences were calculated using one-way ANOVA with Dunnett’s multiple comparisons test against the IRG1 condition for itaconate measurement and against the iNOS condition for nitrite measurement. e, Representative western blot of n = 3 independent experiments showing protein levels of IRG1 and iNOS compared to the loading control GAPDH following co-transfection of HEK cells with IRG1 and iNOS cDNAs. f, Intracellular itaconate and nitrite measured in HEK cells transfected with human IRG1 (ACOD1) cDNA. Data are expressed as the mean of n = 3 independent experiments; error bars, s.e.m. Statistical differences were calculated using a one-way ANOVA with Dunnett’s multiple comparisons test. g, Relative gene expression of ACOD1, NOS2 and GCH1 in human bone marrow organoids compared with GAPDH control. Data are represented in bar charts as the means of n = 3 biological replicates; error bars, s.d. Statistical differences were calculated using a one-way ANOVA with Dunnett’s multiple comparisons test against the CTRL condition. h, Intracellular levels of itaconate in human bone marrow organoids measured by mass spectrometry. Data are represented in bar charts as the mean values of n = 3 biological replicates; error bars, s.d. Statistical differences were calculated using an unpaired t-test. Statistical significance is indicated as ****P < 0.0001; ***P < 0.001; **P < 0.005; *P < 0.05; NS, not significant.Source data