Article Figures & Data
Figures
- Figure 1.
Lactate induces monocyte differentiation into CD14high CD163high macrophages. A, Human monocytes were cultured with GM-CSF and the indicated concentrations of lactate. Results are expressed as a percentage of viable cells at day 5 (n = 5); shaded area, range of lactate concentrations in solid tumors (21). B and C, CD1a, CD14, and CD163 expression was assessed by flow cytometry at day 5 on GM-MΦ, GM+LA-MΦ, M-MΦ, and Mo-DC. Results are expressed as RFI values (n = 5; B) or representative of one of five experiments (C). D, Relative expression of CD14 and CD163 mRNA at day 5 (n = 3). E, The expression of membrane CD163 was evaluated by flow cytometry at the indicated time points (n = 5). A and C–E, Mean ± SEM; **, P < 0.01, ***, P < 0.001 (Wilcoxon test or two-way ANOVA).
- Figure 2.
The effects of lactate are dependent on its internalization and metabolism. A, Human monocytes were generated under acidosis, lactosis, or lactic acidosis, without or with CHC or AZD3965 (AZD). The expression of CD163 and CD14 was assessed by flow cytometry at day 5 (left plots), and IL1β and IL12p70 were quantified after 24-hour stimulation with LPS (right plots; n = 5). B, Monocytes were cultured with GM-CSF and 10 mmol/L 13C-lactate sodium (pH = 6.5); samples were analyzed at day 5 by NMR (left plot, n = 3) and LC-MS (right plot, n = 4). LC-MS, results are expressed as a percentage of 13C isotopic enrichment. A and B, Mean ± SEM; *, P < 0.05; **, P < 0.01; and ***, P < 0.001 (Wilcoxon test).
- Figure 3.
Lactate confers an inflammatory phenotype to human macrophages. A, Day-5 macrophages were stimulated with LPS for 24 hours before quantification of IL1β, IL6, TNFα, CXCL8, IL10, IL12p70, and CXCL10 (n = 7). B, Day-5 GM-MΦ (blue) and GM+LA-MΦ (red) were stimulated for 24 hours with LPS before intracellular staining with anti-TNFα and anti-IL6. Dot plots, gating strategy; histograms, levels of TNFα and IL6 (gray histograms, isotype control mAbs). Results are representative of one of five experiments. FSC, forward scatter; SSC, side scatter. C, Relative expression of IL1β, TNFα, IL6 (top plots), CXCL8, CCL2, CCL13, CXCL5, CXCL2, CXCL3, CXCL1, CCL18, and CCL24 (bottom plots) mRNA by day-5 macrophages either unstimulated (left plot) or stimulated for 6 hours with LPS (right plot; n = 3). A and C, Mean ± SEM; *, P < 0.05; **, P < 0.01; and ***, P < 0.001 (Wilcoxon test).
- Figure 4.
Lactate induces the generation of macrophages with trophic properties. A, Relative HB-EGF, TGFα, EREG, HGF, IL20, OSM, VEGF-A, and PDGF-BB mRNA expression at day 5 (n = 3). Ø, 6-hour stimulation with LPS. B, Day-5 macrophages were stimulated for 24 hours with LPS before quantification of HB-EGF, TGFα, IL20, OSM, VEGF-A, and ET-1 (n = 6). nd, not done. A and B, Mean ± SEM; *, P < 0.05; **, P < 0.01, and ***, P < 0.001 (Wilcoxon test).
- Figure 5.
The expression of selected markers by GM+LA-MΦ is dependent of autocrine M-CSF consumption. A, Relative mRNA expression in day-5 GM-MΦ, GM+LA-MΦ, and M-MΦ (3 different donors) was determined by qPCR. Specific gene expression was calculated using the 2−ΔΔCT method using RPS18, EF1A, TBP, RPL13A, and PPIA as references. The normalized values for each gene are depicted according to a color scale, where red and blue represent expression above and below the mean, respectively. B, GM-MΦ and GM+LA-MΦ were generated without or with GW2580, and the relative expression of SERPINB2, FOLR2, SEPP1, and CD163 mRNA expression was determined at day 5 (n = 5). C, M-CSF quantification in the supernatants collected at indicated time point (n = 5). D, CD115 expression (left plot) and sCD115 production (right plot) were determined at the indicated time points (n = 3). E and F, GM-MΦ and GM+LA-MΦ were generated without or with a neutralizing anti–M-CSF, a control mAb, or GW2580. E, At day 5, the expression of CD163 and CD14 was determined (n = 4). Ab, antibody. F, Day-5 cells were stimulated for 24 hours with LPS and VEGF-A, and TGFα, IL1β, and TNFα were quantified in the supernatants (n = 5). B–F, Mean ± SEM; *, P < 0.05; **, P < 0.01; and ***, P < 0.001 (Wilcoxon test).
- Figure 6.
HIF-1 stabilization and lactate metabolization in GM+LA-MΦ. A, The relative expression of M-CSF and CD115 mRNA was determined in monocytes cultured for 18 hours with GM-CSF or GM-CSF plus lactate, without or with acriflavine (n = 5). B–D, GM-MΦ and GM+LA-MΦ were generated without or with acriflavine. The expression of CD14 and CD163 (B) and the relative expression of CD163, SEPP1, FOLR2, and SERPINB2 mRNA (C) were determined at day 5 (n = 5). D, Day-5 macrophages were stimulated for 24 hours with LPS and IL1β, and TNFα, HB-EGF, and OSM were quantified in cell culture supernatants (n = 5). E, GM+LA-MΦ were generated without or with GSK2837808A and oxamic acid. The expression of CD163 and CD14 was determined at day 5 (left plots), and the production of IL1β and HB-EGF was evaluated after 24-hour stimulation with LPS (right plots; n = 5). *, P < 0.05; **, P < 0.01; and ***, P < 0.001 (Wilcoxon test).
- Figure 7.
Ovarian tumor cell–derived lactate induces the generation of macrophages expressing inflammatory and protumor mediators. Monocytes were cocultured for 5 days with GM-CSF (GM-MΦ) in the absence or presence of the primary ovarian tumor cell CASC170, with or without CHC. A, The expression of CD163 (left plot) and CD14 (middle plot) was evaluated at day 5. Day-5 macrophages were stimulated for 24 hours with LPS before quantification of IL1β (right plot; n = 4). B, The relative expression of SEPP1, FOLR2, STAB1, EREG, and OSM mRNA was assessed after 6-hour stimulation with LPS (n = 4). C, LPS-stimulated or unstimulated cell suspension (after tumor dissociation) before intracellular staining with anti-CXCL8 and anti-IL1β. Dot plots represent the gating strategy and histograms the intracellular expression of CXCL8 and IL1β. Gray histograms, isotype control mAbs. Results are representative of one of three experiments. FSC-A, forward scatter-area; FSC-H, forward scatter-height; FSC-W, forward scatter-width; SSC-A, side scatter-area. D, Relative expression of VEGF in CXCL8− IL1β−, CXCL8+ IL1β−, and CXCL8+ IL1β+ TAMs from 4 patients. A–D, Mean ± SEM; *, P < 0.05; **, P < 0.01; and ***, P < 0.001 (Wilcoxon test).
Volume 8, Issue 3
