Fig. 3

Dexamethasone impaired the glycolytic response to Mtb in human MDM. PBMC were isolated from buffy coats and MDM were adherence purified for 7 days in 10% human serum. MDM were cold lifted and plated on Seahorse XFe24 (Agilent) culture plates (a-f). MDM were treated with VC (ethanol 0.02%) or dexamethasone (0.5 or 5 µM) right before transferring cells into a Seahorse XFe24 Analyzer. After 20 min MDM were stimulated through the ports of the Analyzer with control (a; Seahorse media) or irradiated Mtb (b; iMtb; strain H37Rv, MOI 1–10). ECAR and OCR readings were recorded at regular intervals for 150 min. After 150 min a glycolytic rate assay was performed by injection of Rotenone/Antimycin A followed by 2DG as indicated to calculate GlycoPER from which the induced (c) and compensatory glycolysis (d) were determined. Energy maps were generated from induced glycolysis and the corresponding OCR values (e, f). Cell density was corrected for using Crystal Violet for Seahorse assays (a-f). Alternatively, MDM were treated with VC or dexamethasone for 1 h prior to infection with Mtb for 3 h, MDM were then lysed and RNA extracted (g-j). Expression of PFKFB3 (g), GAPDH (h), PKM2 (i) and ATP5B (j) were assessed by real-time PCR relative to18S. Data are represented as means ± standard deviation. Statistically significant differences were determined using a two-way ANOVA with a Tukey post-test (c, d; n = 7) or Friedman test with Dunn’s post-test (g-j; n = 9); *p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.