Pyruvate Supports RET-Dependent Mitochondrial ROS Production to Control Mycobacterium avium Infection in Human Primary Macrophages

Abstract

Macrophages deploy a variety of antimicrobial programs to contain mycobacterial infection. Upon activation, they undergo extensive metabolic reprogramming to meet an increase in energy demand, but also to support immune effector functions such as secretion of cytokines and antimicrobial activities. Here, we report that mitochondrial import of pyruvate is linked to production of mitochondrial ROS and control of Mycobacterium avium (M. avium) infection in human primary macrophages. Using chemical inhibition, targeted mass spectrometry and single cell image analysis, we showed that macrophages infected with M. avium switch to aerobic glycolysis without any major imbalances in the tricarboxylic acid cycle volume or changes in the energy charge. Instead, we found that pyruvate import contributes to hyperpolarization of mitochondria in infected cells and increases production of mitochondrial reactive oxygen species by the complex I via reverse electron transport, which reduces the macrophage burden of M. avium. While mycobacterial infections are extremely difficult to treat and notoriously resistant to antibiotics, this work stresses out that compounds specifically inducing mitochondrial reactive oxygen species could present themself as valuable adjunct treatments.

Keywords: Mycobacterium avium infection; glycolysis; human primary macrophages; innate immunity; mitochondrial ROS; mitochondrial pyruvate; pyruvate; reverse electron transport.

Figure 1

Glycolysis is required to combat M. avium infection. Human MDMs were challenged for 24 h with 100 ng/ml LPS (blue bars) or infected with M. avium-DsRed (red bars) for 120 min followed by a chase of 24 h (A–C). Glucose (Glc) consumption, lactate (Lac) secretion and glutamine (Gln) consumption were measured using nuclear magnetic resonance. Bar-charts represent average from 5 independent donors. Human MDMs were challenged with 100 ng/ml LPS (blue bars) or infected with M. avium-DsRed (red bars) for 10 min followed by a chase of 24 h (D). Intracellular levels (µM) of glucose-6-phosphate (G6P) and fructose-6-phosphate (F6P) were measured in cell extracts using capillary ion chromatography tandem mass spectrometry. Bar-charts represent the average from 7 independent donors. Human MDMs treated with 2-deoxy-glucose (2-DG) were infected with M. avium-DsRed for 10 min followed by a chase of 72 h (E, F). Intracellular growth was monitored by confocal microscopy. Dots represent the average fluorescence intensity per individual donor (n > 500 cells per donor and per condition), bar-charts represent the average of 7 independent donors. P value between untreated and treated conditions were calculated using the non-parametric ANOVA test (A–D) or using the non-parametric paired test Wilcoxon signed-rank test (F). Scale bars represent 10 µm.

Figure 2

Pyruvate is necessary to maintain mitochondrial hyperpolarization and to control the intracellular burden. Human MDMs were challenged for 24 h with 100 ng/ml LPS (blue bars) or infected with M. avium-DsRed (red bars) for 10 min followed by a chase of 24 h (A). Intracellular levels (µM) of pyruvate were measured in cell extracts using liquid chromatography tandem mass spectrometry. Bar-charts represent the average of 6 independent donors. Human MDMs were treated with various concentration of UK5099 and infected with M. avium-DsRed for 10 min followed by a chase of 72 h (B). Intracellular growth was monitored by confocal microscopy. Dots represent the average fluorescence intensity per individual donor (n > 500 cells per donor and per condition). Bar-charts represent the average of 6 independent donors. Human MDMs were treated with 1.5 µM 2-DG (dark grey) or 100 or 10 µM UK5099 (grey and light grey) and infected with M. avium-DsRed for 10 min followed by a chase of 4 h. Induction of IL-6 (C) and TNF-α (D) expression were tested by real-time PCR. Bar-charts represent average Rq values from 8 independent donors. Human MDMs were challenged for 24 h with 100 ng/ml LPS (blue bars) or infected with M. avium-DsRed (red bars) for 10 min followed by a chase of 24 h (E, F). Intracellular levels (µM) of TCA cycle intermediates (E) and adenine nucleotides (F) were measured in cell extracts using capillary ion chromatography tandem mass spectrometry. Bar-charts represent average values from 7 independent donors. Human MDMs were treated with 10 µM UK5099 and infected with M. avium-DsRed (red) for 10 min followed by a chase of 24 h and mitochondria potential was probed using Mitotracker Green (blue) and DeepRed (green) (G). Merged images are shown. Infected cells are circled in white. Dots represent the average MitoTracker DeepRed/Green fluorescence intensity ratio per individual donor (n > 250 cells per donor and per conditions), bar-charts represent the average of 5 independent donors (H). P value between untreated and treated conditions was calculated using the non-parametric ANAOVA test (A–F) and the non-parametric paired test Wilcoxon signed-rank test (H). Scale bars represent 10 µm.

Figure 3

ROS generated by the complex I is necessary to control intracellular burden. Human MDMs were treated with 10 µM UK5099 and infected with M. avium-CFP for 10 min followed by a chase of 24 h (A, B). Mitochondrial ROS were stained using MitoSOX Red. Merged images are shown. Dots represent the average MitoSOX Red fluorescence intensity for uninfected (white bars) and infected (red bars) cells (n > 250 cells per donor and per condition). Bar-charts represent the average of 6 independent donors. Infected cells are circled in white. Human MDMs were treated with various concentrations of MitoTEMPO (C, red). Intracellular growth was monitored by confocal microscopy. Dots represent the average fluorescence intensity per individual donor (n > 500 cells per donor and per condition), bar-charts represent the average of 5 independent donors. Human MDMs were treated with 10 nM Rotenone (red) and infected with M. avium-CFP for 10 min followed by a chase of 24 h (D). Mitochondrial ROS were stained using MitoSOX Red. Dots represent the average MitoSOX Red fluorescence intensity (n > 250 cell per donor and per condition). Bar-charts represent the average of 6 independent donors. Human MDMs were treated with 10 nM Rotenone, 100 µM Etomoxir or 1 mM DMM and infected with M. avium-DsRed for 10 min followed by a chase of 24 h (E–G, respectively). Intracellular growth was monitored by confocal microscopy. Dots represent the average fluorescence intensity per individual donor (n > 500 cells per donor and per condition), bar-charts represent the average of 7/8 independent donors. P value between untreated (grey) and treated conditions (red) was calculated using the non-parametric ANOVA test (B, C) or the non-parametric paired test Wilcoxon signed-rank test (D–G). Scale bars represent 10 µm. Working model, generated with Biorender (H).