Fig. 4: LDHA^−/− BMDMs exhibit pyruvate-reversible deficiency in their metabolic response to IFNγ.

A ECAR of LDHA^−/− (red) and WT (gray) BMDMs treated with (i) IFNγ (10 ng/mL); (ii) IFNγ and 1 mM pyruvate; and (iii) IFNγ, pyruvate, and 5 µM of the LDH inhibitor GSK 2837808A (darker lines and symbols) relative to untreated BMDMs (lighter lines and symbols). Dashed lines indicate injection of glucose (Glc) followed by oligomycin (OM). Symbols and error bars represent mean ± SEM of 5 biological replicates. B Change in glycolytic capacity of treated BMDMs compared to unstimulated BMDMs (n = 5/group) determined from the profiles in (A). C OCR of BMDMs treated with 10 ng/mL IFNγ (darker lines and symbols) or no stimulus (lighter lines and symbols). Dashed lines indicate injection of OM, FCCP/Pyruvate, or Rotenone/Antimycin A. Symbols and error bars represent mean ± SEM of 5 biological replicates. D Maximal respiration of BMDMs treated as indicated (n = 5/group). E, F Phenograms for the indicated parameters extracted from the XF profiles of BMDMs under the indicated conditions. Symbols and error bars represent the mean ± SEM for 5 biological replicates. G Bacterial burden and (H) NAD⁺:NADH ratio of BMDMs treated as indicated (n = 4/group). I, J Heatmaps representing the abundance of the metabolites comprising key pathways of central carbon metabolism in BMDMs treated with (I) 10 ng/mL IFNγ or (J) 10 ng/mL IFNγ and 1 mM pyruvate. Color-scale values represent the Log₂ fold-change relative to the mean value for WT BMDMs. Upper dendrogram represents hierarchical clustering of replicates based on metabolite abundance. K Abundance (left) and isotopologue distribution (right) of the indicated metabolite and its position within glycolysis (solid line), the PPP (dashed line), and related pathways (dotted lines). Fold change (FC) (left) relative to the mean of WT BMDMs stimulated with IFNγ. Stacked graphs (right) correspond to 100% of the total abundance of each metabolite, where the proportion of each mass isotopologue for each metabolite is indicated by color (n = 4/group). Data are presented as the group mean ± SEM for panels (B, D, G, H, and K) while points represent the individual values for biological (B, D, H, and K) or technical replicates (G). Statistical significance was determined by two-sided, two-sample t-test without assuming equal variance (B, D, G, H) or by two-sided, two-sample Wilcoxon rank-sum test (K). *p < 0.05, **p < 0.01, ****p < 0.0001; exact p-values for each comparison are listed in Supplementary Data 1. Source data are provided as a Source Data file.