Uric acid induces NADPH oxidase-independent neutrophil extracellular trap formation

doi:10.1016/j.bbrc.2013.12.007

Biochemical and Biophysical Research Communications

Volume 443, Issue 2, 10 January 2014, Pages 556-561

https://doi.org/10.1016/j.bbrc.2013.12.007 Get rights and content

Highlights

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Uric acid directly induces neutrophil extracellular trap (NET) formation.
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NET formation induced by uric acid is independent of NADPH oxidase.
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NET formation by uric acid is partially mediated by NF-κB activation.

Abstract

Neutrophil extracellular traps (NETs) are composed of extracellular DNA fibers with antimicrobial peptides that capture and kill microbes. NETs play a critical role in innate host defense and in autoimmune and inflammatory diseases. While the mechanism of NET formation remains unclear, reactive oxygen species (ROS) produced via activation of NADPH oxidase (Nox) are known to be an important requirement. In this study, we investigated the effect of uric acid (UA) on NET formation. UA, a well-known ROS scavenger, was found to suppress Nox-dependent ROS release in a dose-dependent manner. Low concentrations of UA significantly inhibited Nox-dependent NET formation. However, high concentrations of UA unexpectedly induced, rather than inhibited, NET formation. NETs were directly induced by UA alone in a Nox-independent manner, as revealed by experiments using control neutrophils treated with ROS inhibitors or neutrophils of patients with chronic granulomatous disease who have a congenital defect in ROS production. Furthermore, we found that UA-induced NET formation was partially mediated by NF-κB activation. Our study is the first to demonstrate the novel function of UA in NET formation and may provide insight into the management of patients with hyperuricemia.

Introduction

Neutrophils, the first line of defense against the microbes, play a critical role in innate immunity [1]. In infected sites, they phagocytose microbes, degranulate enzymes, and produce reactive oxygen species (ROS) such as superoxide and hydrogen peroxide generated by the NADPH oxidase (Nox) complex. Recently, a novel killing mechanism known as neutrophil extracellular traps (NETs) has been reported. NETs capture microbes with their extracellular structures consisting of DNA fibers and antimicrobial granule proteins [2], [3]. Many physiological stimuli are known to induce NETs. Notably, NET formation is generally ROS-dependent. Patients with chronic granulomatous disease (CGD), who are defective in Nox activity, fail to generate ROS and to make NETs [4]. In a recent study, we demonstrated that singlet oxygen (¹O₂), one species of ROS, is required for Nox-dependent NET formation on stimulation with phorbol myristate acetate (PMA) [5]. Interestingly, neutrophils of CGD patients treated with ¹O₂ in vitro produced NETs, revealing that the pathway could be rescued downstream of Nox [5].

Uric acid (UA), a product of purine metabolism, is a scavenger of ¹O₂ that regulates oxidative stress in humans [6]. Since ¹O₂ is produced by Nox [7], UA is expected to suppress Nox-dependent NET formation. However, a recent study showed that peripheral and synovial fluid neutrophils derived from patients with acute gout, whose UA levels in serum were mostly high, formed NETs [8]. Acute gout is an inflammatory arthritis that is triggered by the deposition of monosodium urate (MSU) crystals, uric acid precipitates in sodium, into the joint space. The inflammatory cascade results in the secretion of inflammatory cytokines, especially interleukin (IL)-1ß and neutrophil recruitment into the joint [9]. Thus, it is still a matter of debate what effect UA directly exerts on NET formation.

In the present study, we first examined the effect of UA on Nox-dependent NET formation by control neutrophils stimulated with PMA. Thereafter, we investigated how UA directly affected NET formation by using control neutrophils treated with ROS inhibitors or CGD neutrophils. Finally, we demonstrate that UA may induce NET formation in a manner distinct from that of PMA.

Section snippets

Reagents

Hanks’ balanced salt solution (HBSS) was purchased from Invitrogen (Carlsbad, CA, USA); trans-1-(2′-methoxyvinyl)pyrene (MVP), Sytox green and orange (for double-strand DNA staining), and dihydrorhodamine 123 (DHR) were ordered from Molecular Probes (Eugene, OR, USA). α-Phenyl-N-tert-butyl nitrone (PBN) was acquired from Radical Research Ltd. (Hino, Tokyo, Japan) and was dissolved in phosphate-buffered saline (PBS) at a final concentration of 100 mM (pH 7.4). Anti-myeloperoxidase (MPO) antibody

Results and discussion

We examined the effect of UA on ¹O₂ production and NET formation by PMA-stimulated neutrophils. First, neutrophils from healthy volunteers were stimulated with PMA with or without UA, and the ¹O₂ production was detected by chemiluminescence. As expected, increasing concentrations of UA (0.05–5 mg/dl) suppressed ¹O₂ production by PMA-stimulated neutrophils in a dose-dependent manner, suggesting that UA is a ¹O₂ scavenger (Fig. 1A and B). Treatments of less than 0.5 mg/dl of UA suppressed

Conflict of interest disclosure

The authors declare no conflict of interest.

Acknowledgments

We thank Keiko Furuta and Haruyasu Kohda (Division of Electron Microscopic Study, Center for Anatomical Studies, Graduate School of Medicine, Kyoto University) for excellent technical assistance. This research was supported by Grants-in-aid for scientific research from the Japan Society for the Promotion of Science (23591474) to K.Y.

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¹: These authors contributed equally to this work.

View full text

Uric acid induces NADPH oxidase-independent neutrophil extracellular trap formation

Highlights

Abstract

Introduction

Section snippets

Reagents

Results and discussion

Conflict of interest disclosure

Acknowledgments

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

J. Pharmacol. Sci.

Hum. Immunol.

J. Cyst. Fibros.

FEBS Lett.

Neutrophils and immunity: challenges and opportunities

Nat. Rev. Immunol.

Neutrophil extracellular traps kill bacteria

Science

Beneficial suicide: why neutrophils die to make NETs

Nat. Rev. Microbiol.

Novel cell death program leads to neutrophil extracellular traps

J. Cell Biol.

Uric acid provides an antioxidant defense in humans against oxidant- and radical-caused aging and cancer: a hypothesis

Proc. Natl. Acad. Sci. U.S.A.

Myeloperoxidase: friend and foe

J. Leukocyte Biol.