Reversed upper glycolysis and rapid activation of oxidative pentose phosphate pathway supports the oxidative burst in neutrophils

Neutrophils are abundant white blood cells at the frontline of innate immunity. Upon stimulation, neutrophils rapidly activate effector functions such as the oxidative burst and neutrophil extracellular traps (NETs) to eliminate pathogens. However, little is known about how neutrophil metabolism powers these functions. Our metabolomic analysis on primary human neutrophils revealed that neutrophil metabolism is rapidly rewired upon pro-inflammatory activation, with particularly profound changes observed in glycolysis and the pentose phosphate pathway (PPP). We found that the stimulation-induced changes in PPP were specifically coupled with the oxidative burst. The oxidative burst requires a large amount of NADPH to fuel superoxide production via NADPH Oxidase (NOX). Isotopic tracing studies revealed that in order to maximize the NADPH yield from glucose metabolism, neutrophils quickly adopt near complete pentose cycle during the oxidative burst. In this metabolic mode, all glucose is shunted into the oxidative PPP, and the resulting pentose-phosphate is recycled back to glucose-6-phosphate, which then re-enters the oxidative PPP. To enable this recycling, net flux through the upper glycolytic enzyme glucose-6-phosphate isomerase (GPI) is completely reversed. This allows oxidative PPP flux in neutrophils to reach greater than two-fold of the glucose uptake rate, far exceeding other known mammalian cells and tissues. Intriguingly, the adoption of this striking metabolic mode is completely dependent on an increased demand for NADPH associated with the oxidative burst, as inhibition of NOX resets stimulated neutrophils to use glycolysis-dominant glucose metabolism, with oxidative PPP flux accounting for less than 10% of glucose metabolism. Together, these data demonstrated that neutrophils have remarkable metabolic flexibility that is essential to enable the rapid activation of their effector functions. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/396838v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@11f9509org.highwire.dtl.DTLVardef@71bffforg.highwire.dtl.DTLVardef@1f402adorg.highwire.dtl.DTLVardef@dbdf1f_HPS_FORMAT_FIGEXP M_FIG C_FIG