Effective NET formation in neutrophils from individuals with G6PD Taiwan-Hakka is associated with enhanced NADP⁺biosynthesis

Abstract

In response to infection, neutrophils employ various strategies to defend against the invading microbes. One of such defense mechanisms is the formation of neutrophil extracellular traps (NETs). Recent studies suggest that reactive oxygen species is a signal critical to NET formation. This prompts us to examine whether neutrophils from individuals with glucose-6-phosphate dehydrogenase (G6PD) Taiwan-Hakka variant, which are prone to oxidative stress generation, have altered ability to form NET. We adopted an image-based method to study the NET formation potential in neutrophils from G6PD-deficient patients. Neutrophils from either normal or G6PD-deficient individuals underwent NETosis in response to phorbol 12-myristate 13-acetate (PMA). The extent of NETosis in the former did not significantly differ from that of the latter. Diphenyleneiodonium sulfate (DPI) and 3-methyladenine (MA) inhibited PMA-stimulated NET formation in these cells, suggesting the involvement of NADPH oxidase and autophagy in the process. Glucose oxidase (GO) and xanthine oxidase/xanthine (XO/X) could induce a similar extent of NET formation in normal and G6PD-deficient neutrophils. GO- or XO-induced NETosis was not inhibitable by MA, implying that reactive oxygen species (ROS) can act as an independent signal for activation of NETosis. Mechanistically, enhanced superoxide production in neutrophils was associated with increases in levels of NAD(+) and NADP(+), as well as activation of NAD(+) kinase. Taken together, these findings suggest that G6PD-deficient neutrophils are as equally efficient as normal cells in NET formation, and their deficiency in G6PD-associated NADPH regeneration capacity is largely compensated for by nicotinamide nucleotide biosynthesis.

Defective nuclear entry of hydrolases prevents neutrophil extracellular trap formation in patients with chronic granulomatous disease

“…To confirm that the classical macroautophagy machinery was not involved in the transport of vesicular NE to the nucleus, we assessed perinuclear co-localization of LC3B and NE in neutrophils isolated from HDs by confocal microscopy. As previously observed (Fig 2), NE translocated to the area around the nucleus 1h post-stimulation with PMA ( Fig In contrast, previous studies had suggested that PI3-kinase inhibition affects NETosis via compromising macroautophagy [3][4][5][6][7][8] . Most of these studies, however, did not analyse the effect of PI3-kinase inhibition on ROS production [4][5][6]8 .…”

“…In order to shed light on the poorly understood steps downstream of ROS, resulting in NETosis, several groups have recently explored macroautophagy [3][4][5][6][7][8] , a cellular degradation pathway for cytoplasmic constituent delivery to lysosomes, which reshapes membrane compartments and could, therefore, be involved in hydrolase access to the nucleus. However, in all of these studies no direct correlation of macroautophagic activity and NETosis could be reported, and only pharmacological inhibition of phosphatidylinositol-3 (PI3)-kinase activity was used to compromise macroautophagy.…”

Glucose-6-Phosphate Dehydrogenase Enhances Antiviral Response through Downregulation of NADPH Sensor HSCARG and Upregulation of NF-κB Signaling

“…For instance, G6PD-deficient granulocytes have an impaired ability to generate superoxide, nitric oxide and hydrogen peroxide [71]. On the other hand, G6PD-deficient neutrophils show increased superoxide and hydrogen peroxide production [72]. More studies are needed to solve such a riddle.…”

Enterovirus 71 Induces Mitochondrial Reactive Oxygen Species Generation That is Required for Efficient Replication

“…Levels of ATP, ADP and AMP were determined using UPLC under conditions as previously described for measurement of nicotinamide nucleotides [41].…”