Physiological and Pathophysiological Roles of Metabolic Pathways for NET Formation and Other Neutrophil Functions

Abstract

Neutrophils are the most numerous cells in the leukocyte population and essential for innate immunity. To limit their effector functions, neutrophils are able to modulate glycolysis and other cellular metabolic pathways. These metabolic pathways are essential not only for energy usage, but also for specialized effector actions, such as the production of reactive oxygen species (ROS), chemotaxis, phagocytosis, degranulation, and the formation of neutrophil extracellular traps (NETs). It has been demonstrated that activated viable neutrophils can produce NETs, which consists of a DNA scaffold able to bind granule proteins and microorganisms. The formation of NETs requires the availability of increased amounts of adenosine triphosphate (ATP) as it is an active cellular and therefore energy-dependent process. In this article, we discuss the glycolytic and other metabolic routes in association with neutrophil functions focusing on their role for building up NETs in the extracellular space. A better understanding of the requirements of metabolic pathways for neutrophil functions may lead to the discovery of molecular targets suitable to develop novel anti-infectious and/or anti-inflammatory drugs.

Keywords: glycolysis; metabolic switch; metabolism; neutrophil; neutrophil extracellular traps.

Figure 1

Overview of key metabolic pathways in neutrophils. In neutrophil cytosol, glycolysis is a primary metabolic route that transforms glucose to pyruvate via a number of enzymes and reactions. Pyruvate is converted to lactate and released out of the cells during anaerobic glycolysis in absence of oxygen. Pyruvate contributes to the TCA cycle after conversion to acetyl-coenzyme A (acetyl Co A) in the presence of oxygen, which results in reducing energy intermediates NADH and dihydroflavine-adenine dinucleotide (FADH2) to generate ATP via the electron transport chain (ETC). Neutrophils also use the pentose-phosphate pathway (PPP) to generate NADPH and riboses, which are then used to build nucleotides, by employing glucose-6-phosphate, a glycolytic pathway intermediate, as an entry point during the oxidative and non-oxidative phases to manufacture NADPH and riboses. NADPH modulates redox signaling and is required for NADPH oxidase-dependent ROS production in neutrophils. Glycogen reserves become concentrated in neutrophils when glucose levels rise, providing glucose-based glycolytic intermediate supply on demand. Through fatty acid synthesis (FAS), the TCA cycle intermediate citrate can be converted to free fatty acids, which can then be transported from the extracellular environment through the FAO pathway to produce acetyl-CoA, which fuels the TCA cycle and produces significantly more energy in the form of ATP. Glutamine helps the TCA cycle by producing α-ketoglutarate through glutaminolysis. Neutrophils also use the glycerol-3-phosphate shuttle to create NAD⁺ from NADH, which aids in mitochondrial membrane potential. HIF-1α augments the activity and expression of GLUT1 and GLUT3, resulting in increased glucose uptake, elevated hexokinase 2 (HK2) and phosphofructokinase B3 (PFKFB3) enzymatic activities, and enhanced ATP production.

Figure 2

Pentose phosphate pathway (PPP). There are two phases in the PPP: oxidative and non-oxidative. G6P-dehydrogenase (G6PD), 6-phosphogluconolactonase, and 6-phosphogluconate dehydrogenase (PGD) convert G6P into CO₂, ribulose-5-phosphate, and NADPH during the oxidative phase to maintain redox balance under stressful conditions. In the non-oxidative phase, enzymes such as ribose-5-phosphate isomerase, ribulose-5-phosphate 3-epimerase, a transketolase, and a transaldolase convert ribulose-5-phosphate to nucleic acids, sugar phosphate precursors, or glycolytic precursors such as fructose-6-phosphate (F6P) and glyceraldehyde-3-phosphate (G3P). PPP and glycolysis share a pool of G3P and F6P, resulting in lactate or pyruvate production.

Figure 3

Glycogen metabolism. Breakdown of glycogen results in glucose-1 phosphate which is then converted to glucose-6-phosphate (G6P) by phosphoglucomutase. G6P can enter the glycolysis pathway and used as a source of energy. Using glucose-6-phosphatase (G6Pase), G6P can be hydrolyzed to glucose via gluconeogenesis and subsequently released into the blood. Glucose-6-phosphate can also be taken by PPP and converted to NADPH or ribose in a variety of tissues.

Figure 4

Glutamine metabolism. Glutamine metabolism results in glutamate, aspartate, lactate and ammonia production. Under physiological conditions glutamine is required for the synthesis of nucleotide precursors, such as RNA and DNA. The sodium-coupled neutral amino acid transporter (SNAT) family of proteins is one of several carrier type transporters (SLCs) responsible for bringing glutamine into the cell. Once inside the cell, glutamine is oxidized and transformed into glutamate. Furthermore, glutamate enters the mitochondria and becomes oxidized and metabolized to α-ketoglutarate, which allows NAD⁺ to be oxygenated and converted to NADH. The TCA cycle may accept α-ketoglutarate and create malate, which is subsequently transformed to pyruvate by the malate dehydrogenase, which oxygenates NADP⁺.

Figure 5

Metabolism of fatty acids. Fatty acids are oxidized to form acetyl-Co A, which is then converted to citrate by the activity of citrate synthase. Acetyl-Co A can be also produced by the mitochondrial matrix and further oxidized in the TCA cycle. Through FAS, the TCA cycle intermediate citrate can be converted to free fatty acids (FFA), which can then be transported from the extracellular environment via the FAO pathway to produce acetyl-Co A, fueling the TCA cycle to produce significantly more energy in the form of ATP.

Figure 6

Bacterial lactic acid generated during infection can induce NET formation. Bacteria-produced lactic acid can cause NET formation in bovine neutrophils, and inhibiting the lactate transporter, the mono-carboxylate or lactate dehydrogenase (LDH) enzyme, prevents this. Because LDH can convert both pyruvate and lactate, it is possible that neutrophils use lactate during NET formation and glycolysis to circumvent the PPP. Furthermore, NET formation in human neutrophils is caused by oxidized low-density lipoprotein (oxLDL) in a ROS-dependent manner upon TLR activation. In addition, non-esterified fatty acids (NEFAs), such as oleic acid (OA) and linoleic acid (LA), initiate FAO, allowing increased ATP production and consequently NET formation.