Metabolomics of PMA-stimulated primary neutrophils

Since metabolic activity affects the oxidative burst and NET formation, I investigated the metabolomic dynamics during NET formation.

I prepared neutrophil samples of 10 healthy donors at different time points of early NET formation, i.e. 15, 30 and 45 minutes after PMA stimulation (Figure 14). Mass spectrometry-based global metabolite measurement and subsequent analysis was performed externally by Metabolon, Inc. Metabolites of selected metabolism pathways are presented below.

Figure 14 | Experimental set-up for PMA-induced neutrophil metabolomic measurements.

10⁷ cells were distributed in 3.5 ml medium and stimulated with 100 nM PMA for 15-45 minutes.

Subsequently, cells were washed and the cell pellet shock frozen and stored until shipment.

3.3.1 Glycolysis and Pentose Phosphate Pathway

Glycolysis and the PPP are part of the central cellular metabolism. Metabolome analysis revealed a statistically significant mild increase of glucose which lies at the very beginning of the glycolysis pathway 30 and 45 minutes after PMA stimulation (Figure 15 A). Interestingly, glucose-6-phosphate which is the metabolite immediately following uptake of glucose into the cell was not detected in the global metabolomic measurement (not shown).

3-phosphoglycerate is a metabolite further downstream during glycolysis and showed a significant increase at all three time points (Figure 15 B). Similarly, the abundance of phosphoenolpyruvate (PEP) which lies at the end of the glycolytic pathway was significantly higher at all three time points (Figure 15 C).

Figure 15 | Abundance of the glycolysis metabolites glucose, 3-phosphoglycerate and phosphoenolpyruvate significantly increases over time in PMA-stimulated neutrophils.

The box plot line indicates the median value and the cross indicates the mean value. Statistical significance was determined performing one-way ANOVA with repeated measurements. A, Glucose concentration is significantly increased at time points 30 and 45 minutes. B, The 3-phosphoglycerate concentration is significantly higher at all three time points. C, The phosphoenolpyruvate (PEP) concentration is significantly higher at all three time points.

6-phosphogluconate and sedoheptulose-7-phosphate are intermediate metabolites of the oxidative and non-oxidative PPP, respectively. Both show an increase upon PMA stimulation (Figure 16) indicating increased PPP activity. Whereas the increase in 6-PG reaches statistical significance after already 15 minutes and stays high throughout the measured time span (Figure 16 A), S7P reaches a significant level of increase only after 45 minutes (Figure 16 B).

Figure 16 | Abundance of the pentose phosphate pathway metabolites 6-phosphogluconate and sedoheptulose-7-phosphate increases over time compared to the control cells.

The box plot line indicates the median value and the cross indicates the mean value. Statistical significance was determined performing one-way ANOVA with repeated measurements. A, the concentration of 6-phosphogluconate is significantly higher at all three time points. B, the sedoheptulose-7-phosphate concentration is significantly higher after 45 minutes.

3.3.2 Redox Balance

Maintaining a stable redox balance is crucial for cellular homeostasis. PMA stimulation and NET formation go hand in hand with a rapid and massive production of ROS in neutrophils.

The glutathione redox system plays an important role in keeping the cellular redox balance.

Surprisingly, the amount of GSH remained unaltered throughout all time points (Figure 17 A) whereas GSSG significantly decreased after 30 minutes (Figure 17 B). Interestingly, the degradation product of GSH, cysteinylglycine, was significantly reduced after 30 and 45 minutes (Figure 17 C).

Methionine sulfoxide, an early precursor molecule of GSH is significantly increased already after 15 minutes of PMA stimulation and decreases subsequently, although it remains significantly above the baseline level (Figure 17 D).

Figure 17 | Abundance of compounds in the oxidative stress and redox balance pathway.

The box plot line indicates the median value and the cross indicates the mean value. Statistical significance was determined performing one-way ANOVA with repeated measurements. A, the concentration of reduced GSH is not significantly altered at any time point. B, the concentration of GSSG is significantly reduced after 30 minutes and shows a decreasing trend after 45 minutes. C, the concentration of cysteinylglycine is significantly reduced after 30 and 45 minutes. D, the methionine sulfoxide concentration is significantly increased at all three time points.

3.3.3 Nucleotide metabolism

Metabolites of the nucleotide metabolism were also included in the global metabolome analysis. Adenosine 3-monophosphate (3-AMP) abundance is significantly increased 15 minutes after PMA stimulation and subsequently returns to baseline levels (Figure 18 A).

Other nucleotide precursors on the contrary, such as guanine, uracil and guanosine were significantly reduced in their concentration over the course of 45 minutes PMA stimulation (Figure 18 B-D).

Figure 18 | Abundance of compounds in the nucleotide metabolism pathway are significantly altered in PMA-stimulated neutrophils.

The box plot line indicates the median value and the cross indicates the mean value. Statistical significance was determined performing one-way ANOVA with repeated measurements. A, the concentration of adenosine 3’-monophosphate (3’-AMP) is significantly increased after 15 minutes and returns to baseline thereafter. B, the concentration of guanine is significantly reduced after 30 and after 45 minutes. C, the concentration of uracil is significantly reduced after 30 and 45 minutes. D, the guanosine concentration is significantly reduced at all three time points.

3.3.4 Phospholipid metabolism

Phospholipids are the building stones of cellular membranes and play a role in cellular signalling. Choline phosphate, choline and glycerophosphoethanolamine underwent the highest concentration changes among the phospholipids. Whereas choline phosphate was steadily and significantly reduced over the 45 minutes to only 30 % of the baseline abundance, the choline concentration went the opposite way and tripled already after 15 minutes without subsequent further increases (Figure 19 A & B).

Glycerophosphoethanolamine increased significantly over the time course and more than quadrupled after 15 minutes before reaching more than 8 and more than 10 times the base line level after 30 and 45 minutes, respectively (Figure 19 C).

Figure 19 | Abundance of compounds in the phospholipid metabolism.

The box plot line indicates the median value and the cross indicates the mean value. Statistical significance was determined performing one-way ANOVA with repeated measurements. A, the concentration of choline phosphate is significantly decreased after 15, 30 and 45 minutes. B, the concentration of choline is significantly increased after 15, 30 and 45 minutes. C, the concentration of glycerophosphoethanolamine is very significantly increased at all three time points.