Hyperoxidation of ether-linked phospholipids accelerates neutrophil extracellular trap formation

Scientific Reports, Nov 2017

Because neutrophil extracellular trap (NET) formation is involved in the pathology of a wide variety of diseases, NET-regulating compounds are expected to be useful for the therapies of these diseases. In this study, we identified sulfasalazine (SSZ) as a potent enhancer of NET formation both in vitro and in vivo. Although SSZ did not increase the amount of ROS generated, it accelerated the generation of ether-linked oxidized phospholipids, such as PE (18;1e/15-HETE) and PC (16;0e/13-HODE). Trolox, but not 2-ME, effectively suppressed lipid oxidation and NET formation that were induced by SSZ. SSZ is known as a potent inducer of ferroptosis in cancer cells by inhibiting xCT, a component of the cystine transporter. However, we found that SSZ accelerated NET formation in an xCT-independent manner. Structure-activity relationship studies revealed that the sulfapyridine moiety of SSZ plays a central role in enhancing NET formation. Furthermore, we found that two additional sulfonamide and sulfone derivatives possess NET-inducing activity by accelerating lipid oxidation. These results indicate that the hyperoxidation of ether-linked phospholipids is a key mechanism for accelerating NET formation.

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Hyperoxidation of ether-linked phospholipids accelerates neutrophil extracellular trap formation

Abstract Because neutrophil extracellular trap (NET) formation is involved in the pathology of a wide variety of diseases, NET-regulating compounds are expected to be useful for the therapies of these diseases. In this study, we identified sulfasalazine (SSZ) as a potent enhancer of NET formation both in vitro and in vivo. Although SSZ did not increase the amount of ROS generated, it accelerated the generation of ether-linked oxidized phospholipids, such as PE (18;1e/15-HETE) and PC (16;0e/13-HODE). Trolox, but not 2-ME, effectively suppressed lipid oxidation and NET formation that were induced by SSZ. SSZ is known as a potent inducer of ferroptosis in cancer cells by inhibiting xCT, a component of the cystine transporter. However, we found that SSZ accelerated NET formation in an xCT-independent manner. Structure-activity relationship studies revealed that the sulfapyridine moiety of SSZ plays a central role in enhancing NET formation. Furthermore, we found that two additional sulfonamide and sulfone derivatives possess NET-inducing activity by accelerating lipid oxidation. These results indicate that the hyperoxidation of ether-linked phospholipids is a key mechanism for accelerating NET formation. Introduction Polymorphonuclear leukocytes, or neutrophils, are sentinel cells that are part of the first line of defense against bacterial infection. These cells contribute to eliminating invading bacteria via cell killing and phagocytic activity. Recently, another mechanism has been identified as a novel defense mechanism used by neutrophils1,2. Activated neutrophils undergo a distinct type of cell death called NETosis that is morphologically distinct from both apoptosis and necrosis3. This cell death process has been associated with the extracellular release of chromosomal DNA that is coated with histones and proteases and that forms web-like structures4. These structures are named neutrophil extracellular traps (NETs), and they play critical roles in the efficient elimination of bacteria by immobilizing bacterial cells. Although NET formation is generally considered to be a consequence of the NETosis cell death process, NET formation was recently found to occur without cellular suicide5,6. NETs are generated in response to a variety of stimuli, including bacterial components, monosodium urate, cholesterol crystals, and pharmacological reagents, such as phorbol-12-myristate-13 acetate (PMA) and ionomycin. In response to these stimuli, reactive oxygen species (ROS) are generated in an NADPH oxidase-dependent manner. The resulting oxidative stress can induce the activation of protein-arginine deiminase 4 (PAD4)7,8. PAD4 is specifically expressed by neutrophils, and activated PAD4 mediates the citrullination of histone H3 in N-terminal arginine residues, which results in the decondensation of DNA and NET formation7,9,10. Inhibiting PAD4 activity using chemical inhibitors suppressed NET formation in mouse and human neutrophils11,12. PAD4-deficient mice exhibited impaired NET formation and susceptibility to bacterial infection, demonstrating that PAD4 is important during NET formation8. However, little is currently known about the signaling pathways that lie downstream of the ROS for PAD4 activation. It has also been reported that neutrophil elastase (NE) is implicated in efficient NET formation. NE is translocated from granules into the nucleus in an ROS- and myeloperoxidase-dependent manner. Once in the nucleus, NE cleaves histones to promote chromatin decondensation13,14. In addition to the chromatin decondensation induced by PAD4 and NE, the nuclear membrane must also be disrupted for NET formation to occur. However, the precise mechanisms underlying each of these events remain obscure. NET formation has recently attracted a great deal of attention not only as a defense mechanism but also because it is implicated in the pathologies underlying a wide variety of diseases1,15. During NETosis, dying neutrophils may release intracellular materials, including damage-associated molecular patterns (DAMPs), which can cause the deterioration of tissue injuries or delay regeneration1. For instance, neutrophils obtained from diabetic humans and mice are more susceptible to NETosis and, as a consequence, wound healing is delayed in diabetic mice16. In individuals with ischemic reperfusion injury, NET formation is mediated by TLR signaling and exacerbates sterile injury17. On the other hand, there are some diseases in which NET formation has beneficial effects on pathology. For example, neutrophils undergo NETosis, leading to NET formation at inflamed sites in gout, and the formation of NET leads to the resolution of inflammation by degrading inflammatory cytokines18. Furthermore, neutrophils detect human immunodeficiency virus (HIV)-1 by Toll-like receptors (TLRs) 7 and 8, which recognize viral nucleic acids. Engagement of TLRs 7 and 8 induces NET formation, leading to NET-dependent HIV-1 elimination19. Because NE (...truncated)


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Satoshi Yotsumoto, Yuito Muroi, Tatsuya Chiba, Rio Ohmura, Maki Yoneyama, Megumi Magarisawa, Kosuke Dodo, Naoki Terayama, Mikiko Sodeoka, Ryohei Aoyagi, Makoto Arita, Satoko Arakawa, Shigeomi Shimizu, Masato Tanaka. Hyperoxidation of ether-linked phospholipids accelerates neutrophil extracellular trap formation, Scientific Reports, 2017, Issue: 7, DOI: 10.1038/s41598-017-15668-z