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. 2018 Jul 6;9:1552. doi: 10.3389/fimmu.2018.01552

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Copyright © 2018 Ohl, Fragoulis, Klemm, Baumeister, Klock, Verjans, Böll, Möllmann, Lehrke, Costa, Denecke, Schippers, Roth, Wagner, Wruck and Tenbrock.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Nrf2 induces metabolic activity in myeloid-derived suppressor cells (MDSCs). (A) Metabolic enzymes regulated by Nrf2 in glucose metabolism in MDSCs (own microarray and RT-qPCR data). (B) N-fold mRNA expression of metabolic enzymes in MACS isolated Keap^fl/fl and VAV^creKeap^fl/fl CD11b⁺Gr-1⁺ (n = 7–8, PFKFB3 n = 5) cells analyzed by RT-qPCR. Bars indicate mean and error bars SEM, two-tailed one sample test. (C) 2NBD-glucose incorporation was analyzed in CD11b⁺Gr1⁺ splenocytes from Keap^fl/fl (n = 3) and VAV^creKeap^fl/fl (n = 4) mice at different time-points, two-tailed unpaired t-test. (D) BM-derived cells were incubated with GM-CSF and IL-6 for 8 days with different glucose concentrations, and frequencies of CD11b⁺Gr-1⁺ cells were assessed by flow cytometry. Bars indicate mean ± SEM of three independent experiments per group, two-tailed unpaired t-test. (E,F) BM-derived cells were incubated with GM-CSF and IL-6 for 8 days in the absence or presence of 1 µM rapamycin, n = 4, two-tailed unpaired t-test. (G) Representative histogram of five experiments (p = 0.0155, two-tailed paired t-test) showing p-mTOR expression in splenic CD11b⁺Gr-1⁺ cells. (H) Mean fluorescent intensity of pS6 expression in CD11b⁺Gr-1⁺ cells. (I) Statistical analysis of mitochondrial mass of Keap^fl/fl (n = 4) and VAV^creKeap^fl/fl (n = 4) CD11b⁺Gr-1⁺ cells evaluated by assessing NAO mean fluorescence intensity, n = 3, two-tailed unpaired t-test. (J) OCR measured under basal conditions and after addition of the indicated drugs. Points indicate mean from six Keap^fl/fl (n = 6) and six VAV^creKeap^fl/fl (n = 6) CD11b⁺Gr-1⁺ cells, error bars SEM. (K) Statistical analysis of max. OCR. Bars indicate mean ± SEM. (L) ECAR measured under basal conditions and after addition of the indicated drugs. Points indicate mean from six Keap^fl/fl (n = 6) and six VAV^creKeap^fl/fl (n = 6) CD11b⁺Gr-1⁺ cells, error bars SEM, two-tailed unpaired t-test.

Nrf2 induces metabolic activity in myeloid-derived suppressor cells (MDSCs). (A) Metabolic enzymes regulated by Nrf2 in glucose metabolism in MDSCs (own microarray and RT-qPCR data). (B) N-fold mRNA expression of metabolic enzymes in MACS isolated Keap^fl/fl and VAV^creKeap^fl/fl CD11b⁺Gr-1⁺ (n = 7–8, PFKFB3 n = 5) cells analyzed by RT-qPCR. Bars indicate mean and error bars SEM, two-tailed one sample test. (C) 2NBD-glucose incorporation was analyzed in CD11b⁺Gr1⁺ splenocytes from Keap^fl/fl (n = 3) and VAV^creKeap^fl/fl (n = 4) mice at different time-points, two-tailed unpaired t-test. (D) BM-derived cells were incubated with GM-CSF and IL-6 for 8 days with different glucose concentrations, and frequencies of CD11b⁺Gr-1⁺ cells were assessed by flow cytometry. Bars indicate mean ± SEM of three independent experiments per group, two-tailed unpaired t-test. (E,F) BM-derived cells were incubated with GM-CSF and IL-6 for 8 days in the absence or presence of 1 µM rapamycin, n = 4, two-tailed unpaired t-test. (G) Representative histogram of five experiments (p = 0.0155, two-tailed paired t-test) showing p-mTOR expression in splenic CD11b⁺Gr-1⁺ cells. (H) Mean fluorescent intensity of pS6 expression in CD11b⁺Gr-1⁺ cells. (I) Statistical analysis of mitochondrial mass of Keap^fl/fl (n = 4) and VAV^creKeap^fl/fl (n = 4) CD11b⁺Gr-1⁺ cells evaluated by assessing NAO mean fluorescence intensity, n = 3, two-tailed unpaired t-test. (J) OCR measured under basal conditions and after addition of the indicated drugs. Points indicate mean from six Keap^fl/fl (n = 6) and six VAV^creKeap^fl/fl (n = 6) CD11b⁺Gr-1⁺ cells, error bars SEM. (K) Statistical analysis of max. OCR. Bars indicate mean ± SEM. (L) ECAR measured under basal conditions and after addition of the indicated drugs. Points indicate mean from six Keap^fl/fl (n = 6) and six VAV^creKeap^fl/fl (n = 6) CD11b⁺Gr-1⁺ cells, error bars SEM, two-tailed unpaired t-test.