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. 2017 Nov 7;7:23–34. doi: 10.1016/j.molmet.2017.11.002

Figure 1.

Figure 1

OxPLs induce a metabolic shift to glutathione synthesis in macrophages. A. mRNA expression by qPCR measured in BMDMs treated with 1 μg/mL LTA (M1), 10 ng/mL IL4 (M2), or 10 μg/mL OxPAPC (Mox) for 4 h. Genes measured are Nos2 (iNOS; M1 marker), Arg1 (M2 marker), and Ho1 (Mox marker) (n ≥ 3). B. Metabolomics was performed on bone marrow-derived macrophages (BMDMs; harvested and cultured from C57BL/6 mouse hind legs) after 6 h treatment with M0 (vehicle, RPMI media), M1 (1 μg/mL LTA), M2 (10 ng/mL IL4), and Mox (10 μg/mL OxPAPC) stimuli (n = 5). Of a total of 512 metabolites of known identity, following normalization to protein concentration by Bradford assay, Welch's two-sample t-test was used to identify metabolites that differed significantly between experimental groups. A summary of the numbers of metabolites that achieved statistical significance (Welch's 2-sided t-test, p ≤ 0.05) either uniquely per individual treatment or commonly between treatments, represented in a Venn diagram (n = 5). C. Volcano plots produced using data from metabolomics of polarized BMDMs (see A) highlighting significantly (p ≤ 0.05) increased (red) or decreased (green) metabolites per group. Metabolites related to energy metabolism and redox homeostasis significantly changed in Mox (OxPAPC-treated) BMDMs are highlighted as red circles in each panel. Ornithine is highlighted in the M2 (IL4-treated) BMDMs as a positive control. D. Quantification of individual metabolites involved in the glutathione synthesis pathway in BMDMs represented by box and whisker plots. The highest and lowest bars represent the maximum distribution, while the upper, middle, and lower lines of the box represent the first quartile, median, and third quartile, respectively (n = 5). E. Glutathione synthesis pathway in Mox BMDMs (10 μg/mL OxPAPC, 6 h). #GSH (reduced glutathione) and GSSG (oxidized glutathione) levels were not measured. F. Intracellular reduced glutathione (GSH) levels in BMDMs treated for 6 or 24 h with vehicle (RPMI media) or 10 μg/mL OxPAPC, measured using the GSH/GSSG Ratio Detection Assay Kit (Abcam; ab138881) (n = 4). Data are expressed as mean ± SEM. Biological replicates indicated by (n). Statistical significance calculated by Welch's 2-sided t-test (*p ≤ 0.05; **p < 0.01; ***p < 0.001).