Rab27a Is Essential for the Formation of Neutrophil Extracellular Traps (NETs) in Neutrophil-Like Differentiated HL60 Cells

Neutrophils play a crucial role in host defence. In response to a variety of inflammatory stimulation, they form neutrophil extracellular traps (NETs). NETs are extracellular structures composed of chromatin fibers decorated with antimicrobial proteins and developing studies indicate that NETs contribute to extracellular microbial killing. While the intracellular signaling pathways that regulate NET formation remain largely unknown, there is growing evidence that generation of reactive oxygen species (ROS) is a key event for NET formation. The Rab family small GTPase Rab27a is an important component of the secretory machinery of azurophilic granules in neutrophils. However, the precise mechanism of NET formation and whether or not Rab27a contributes to this process are unknown. Using neutrophil-like differentiated HL60 cells, we show here that Rab27a plays an essential role in both phorbol myristate acetate (PMA)- and Candida albicans-induced NET formation by regulating ROS production. Rab27a-knockdown inhibited ROS-positive phagosome formation during complement-mediated phagocytosis. To investigate the role of Rab27a in neutrophil function in detail, both primary human neutrophils and neutrophil-like differentiated HL60 cells were treated with PMA, and NET formation process was assessed by measurement of release of histone H3 into the medium, citrullination of the arginine in position 3 of histone H4 and chase of the nuclear change of the living cells in the co-existence of both cell-permeable and -impermeable nuclear indicators. PMA-induced NET formation occured sequentially in both neutrophil-like differentiated HL60 cells and primary neutrophils, and Rab27a-knockdown clearly inhibited NET formation in association with reduced ROS production. We also found that serum-treated Candida albicans triggers NET formation in a ROS-dependent manner, and that Rab27a-knockdown inhibits this process as well. Our findings demonstrate that Rab27a plays an important role in NET formation induced by both Candida albicans infection and PMA treatment by regulating ROS production.     

Impaired neutrophil extracellular trap formation: a novel defect in the innate immune system of aged individuals

Neutrophil extracellular traps (NETs) are a recently discovered addition to the defensive armamentarium of neutrophils, assisting in the immune response against rapidly dividing bacteria. Although older adults are more susceptible to such infections, no study has examined whether aging in humans influences NET formation. We report that TNF‐α‐primed neutrophils generate significantly more NETs than unprimed neutrophils and that lipopolysaccharide (LPS)‐ and interleukin‐8 (IL‐8)‐induced NET formation exhibits a significant age‐related decline. NET formation requires generation of reactive oxygen species (ROS), and this was also reduced in neutrophils from older donors identifying a mechanism for reduced NET formation. Expression of IL‐8 receptors (CXCR1 and CXCR2) and the LPS receptor TLR4 was similar on neutrophils from young and old subjects, and neutrophils challenged with phorbol‐12‐myristate‐13‐acetate (PMA) showed no age‐associated differences in ROS or NET production. Taken together, these data suggest a defect in proximal signalling underlies the age‐related decline in NET and ROS generation. TNF‐α priming involves signalling through p38 MAP kinase, but activation kinetics were comparable in neutrophils from young and old donors. In a clinical setting, we assessed the capacity of neutrophils from young and older patients with chronic periodontitis to generate NETs in response to PMA and hypochlorous acid (HOCL). Neutrophil extracellular trap generation to HOCL, but not PMA, was lower in older periodontitis patients but not in comparison with age‐matched controls. Impaired NET formation is thus a novel defect of innate immunity in older adults but does not appear to contribute to the increased incidence of periodontitis in older adults.     

Uric acid induces NADPH oxidase-independent neutrophil extracellular trap formation

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.     

Reactive oxygen species‐induced activation of ERK and p38 MAPK mediates PMA‐induced NETs release from human neutrophils

Neutrophils/polymorphonuclear leukocytes (PMNs), an important component of innate immune system, release extracellular traps (NETs) to eliminate invaded pathogens; however understanding of the role of signaling molecules/proteins need to be elucidated. In the present study role of p38 MAPK and extracellular signal regulated kinase (ERK) against phorbol 12‐myristate 13‐acetate (PMA) induced reactive oxygen species (ROS) generation and NETs formation has been investigated. Human neutrophils were treated with PMA to induce free radical generation and NETs release, which were monitored by NBT reduction and elastase/DNA release, respectively. PMA treatment led to the time dependent phosphorylation of p38 MAPK and ERK in PMNs. Pretreatment of PMNs with SB202190 or U0126 did not significantly reduce PMA induce free radical generation, but prevented NETs release. Pretreatment of PMNs with NADPH oxidase inhibitor (diphenyleneiodonium chloride) significantly reduced free radical generation, p38 MAPK and ERK phosphorylation as well as NETs release, suggesting that p38 MAPK and ERK activation was downstream to free radical generation. The present study thus demonstrates ROS dependent activation of ERK and p38 MAPK, which mediated PMA induced NETs release from human neutrophils. J. Cell. Biochem. 114: 532–540, 2013. © 2012 Wiley Periodicals, Inc.     

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.     

The Extracellular Matrix of Candida albicans Biofilms Impairs Formation of Neutrophil Extracellular Traps

Neutrophils release extracellular traps (NETs) in response to planktonic C. albicans. These complexes composed of DNA, histones, and proteins inhibit Candida growth and dissemination. Considering the resilience of Candida biofilms to host defenses, we examined the neutrophil response to C. albicans during biofilm growth. In contrast to planktonic C. albicans, biofilms triggered negligible release of NETs. Time lapse imaging confirmed the impairment in NET release and revealed neutrophils adhering to hyphae and migrating on the biofilm. NET inhibition depended on an intact extracellular biofilm matrix as physical or genetic disruption of this component resulted in NET release. Biofilm inhibition of NETosis could not be overcome by protein kinase C activation via phorbol myristate acetate (PMA) and was associated with suppression of neutrophil reactive oxygen species (ROS) production. The degree of impaired NET release correlated with resistance to neutrophil attack. The clinical relevance of the role for extracellular matrix in diminishing NET production was corroborated in vivo using a rat catheter model. The C. albicans pmr1Δ/Δ, defective in production of matrix mannan, appeared to elicit a greater abundance of NETs by scanning electron microscopy imaging, which correlated with a decreased fungal burden. Together, these findings show that C. albicans biofilms impair neutrophil response through an inhibitory pathway induced by the extracellular matrix.     

Vitamin D metabolites and analogs induce lipoxygenase mRNA expression and activity as well as reactive oxygen species (ROS) production in human bone cell line

Vitamin D metabolites and its less-calcemic analogs (vitamin D compounds) are beneficial for bone and modulate cell growth and energy metabolism. We now analyze whether 25(OH)D(3) (25D), 1,25(OH)(2)D(3) (1,25D), 24,25(OH)(2)D(3) (24,25D), JKF1624F(2)-2 (JKF) or QW1624F(2)-2 (QW) regulate lipooxygenase (LO) mRNA expression and its products; hydroxyl-eicosatetraenoic acid (12 and 15HETE) formation, as well as reactive oxygen species (ROS) production in human bone cell line (SaOS2) and their interplay with modulation of cell proliferation and energy metabolism. All compounds except 25D increased 12LO mRNA expression and modulated 12 and 15HETE production whereas ROS production was increased by all compounds, and inhibited by NADPH oxidase inhibitors diphenyleneiodonium (DPI) and N-acetylcysteine (NAc). Baicaleine (baic) the inhibitor of 12 and 15LO activity blocked only slightly the stimulation of DNA synthesis by all compounds, whereas DPI inhibited almost completely the stimulation of DNA and CK by all compounds. Treatments of cells with 12 or 15HETE increased DNA synthesis and CK that were only slightly inhibited by DPI. These results indicate that vitamin D compounds increased oxidative stress in osteoblasts in part via induction of LO expression and activity. The increased ROS production mediates partially elevated cell proliferation and energy metabolism, whereas the LO mediation is not essential. This new feature of vitamin D compounds is mediated by intracellular and/or membranal binding sites and its potential hazard could lead to damage due to increased lipid oxidation, although the transient mediation of ROS in cell proliferation is beneficial to bone growth in a yet unknown mechanism.     

Respiratory Syncytial Virus Fusion Protein Promotes TLR-4–Dependent Neutrophil Extracellular Trap Formation by Human Neutrophils

Acute viral bronchiolitis by Respiratory Syncytial Virus (RSV) is the most common respiratory illness in children in the first year of life. RSV bronchiolitis generates large numbers of hospitalizations and an important burden to health systems. Neutrophils and their products are present in the airways of RSV-infected patients who developed increased lung disease. Neutrophil Extracellular Traps (NETs) are formed by the release of granular and nuclear contents of neutrophils in the extracellular space in response to different stimuli and recent studies have proposed a role for NETs in viral infections. In this study, we show that RSV particles and RSV Fusion protein were both capable of inducing NET formation by human neutrophils. Moreover, we analyzed the mechanisms involved in RSV Fusion protein-induced NET formation. RSV F protein was able to induce NET release in a concentration-dependent fashion with both neutrophil elastase and myeloperoxidase expressed on DNA fibers and F protein-induced NETs was dismantled by DNase treatment, confirming that their backbone is chromatin. This viral protein caused the release of extracellular DNA dependent on TLR-4 activation, NADPH Oxidase-derived ROS production and ERK and p38 MAPK phosphorylation. Together, these results demonstrate a coordinated signaling pathway activated by F protein that led to NET production. The massive production of NETs in RSV infection could aggravate the inflammatory symptoms of the infection in young children and babies. We propose that targeting the binding of TLR-4 by F protein could potentially lead to novel therapeutic approaches to help control RSV-induced inflammatory consequences and pathology of viral bronchiolitis.     

Induction of reactive oxygen species from isolated rat glomeruli by protein kinase C activation and TNF-alpha stimulation, and effects of a phosphodiesterase inhibitor

Diabetic nephropathy is a major complication of diabetes leading to end-stage renal disease, which requires hemodialysis. Although the mechanism by which it progresses is largely unknown, the role of hyperglycemia-derived oxidative stress has recently been the focus of attention as the cause of diabetic complications. Constituent cells of the renal glomeruli have the capacity to release reactive oxygen species (ROS) upon stimulation of NADPH oxidase activated by protein kinase C (PKC). Hyperglycemia and insulin resistance in the diabetic state are often associated with activation of PKC and tumor necrosis factor (TNF)-alpha, respectively. The aim of this study is to clarify the signaling pathway leading to ROS production by PKC and TNF-alpha in rat glomeruli. Isolated rat glomeruli were stimulated with phorbol 12-myristate 13-acetate (PMA) and TNF-alpha, and the amount of ROS was measured using a chemiluminescence method. Stimulation with PMA (10 ng/ml) generated ROS with a peak value of 136+/-1.2 cpm/mg protein (mean+/-SEM). The PKC inhibitor H-7, the NADPH oxidase inhibitor diphenylene iodonium and the phosphatidylinositol-3 (PI-3) kinase inhibitor wortmannin inhibited PMA-induced ROS production by 100%, 100% and 80%, respectively. In addition, TNF-alpha stimulated ROS production (283+/-5.8/mg protein/20 min). The phosphodiesterase inhibitor cilostazol activates protein kinase A and is reported to improve albuminuria in diabetic rats. Cilostazol (100 microg/ml) inhibited PMA, and TNF-alpha-induced ROS production by 78+/-1.8, and 19+/-2.7%, respectively. The effects of cilostazol were not additive with wortmannin. Cilostazol arrests oxidative stress induced by PKC activation by inhibiting the PI-3 kinase-dependent pathway, and may thus prevent the development of diabetic nephropathy.     

Quantitative analysis of hemin-induced neutrophil extracellular trap formation and effects of hydrogen peroxide on this phenomenon

Formation of neutrophil extracellular traps (NETs) can perpetuate sterile inflammation; thus, it is important to clarify their pathophysiological characteristics. Free heme, derived via hemolysis, is a major contributor to organ damage, and reportedly induces neutrophil activation as well as reactive oxygen species (ROS) production and NET formation. For this study, we examined hemin (Fe³⁺ -protoporphyrin IX)-induced NET formation quantitatively in vitro as well as the effects of oxidative stress.NETs formed in vitro from cultured neutrophils were quantitatively detected by using nuclease treatment and Sytox Green, a nucleic acid stain. Hemin-induced NET production was found to be in a dose-dependent manner, NADPH oxidase-dependent and toll-like receptor (TLR)-4 independent. Additionally, the iron molecule in the porphyrin ring was considered essential for the formation of NETs. In the presence of low concentrations of hydrogen peroxide, low concentrations of hemin-induced NETs were enhanced, unlike those of phorbol myristate acetate (PMA)-induced NETs.Quantitative analysis of NET formation may prove to be a useful tool for investigating NET physiology, and hemin could function as a possible therapeutic target for hemolysis-related events.     

A Metabolic Shift toward Pentose Phosphate Pathway Is Necessary for Amyloid Fibril- and Phorbol 12-Myristate 13-Acetate-induced Neutrophil Extracellular Trap (NET) Formation

Neutrophils are the main defense cells of the innate immune system. Upon stimulation, neutrophils release their chromosomal DNA to trap and kill microorganisms and inhibit their dissemination. These chromatin traps are termed neutrophil extracellular traps (NETs) and are decorated with granular and cytoplasm proteins. NET release can be induced by several microorganism membrane components, phorbol 12-myristate 13-acetate as well as by amyloid fibrils, insoluble proteinaceous molecules associated with more than 40 different pathologies among other stimuli. The intracellular signaling involved in NET formation is complex and remains unclear for most tested stimuli. Herein we demonstrate that a metabolic shift toward the pentose phosphate pathway (PPP) is necessary for NET release because glucose-6-phosphate dehydrogenase (G6PD), an important enzyme from PPP, fuels NADPH oxidase with NADPH to produce superoxide and thus induce NETs. In addition, we observed that mitochondrial reactive oxygen species, which are NADPH-independent, are not effective in producing NETs. These data shed new light on how the PPP and glucose metabolism contributes to NET formation.     

Diphenyleneiodonium induces ROS-independent p53 expression and apoptosis in human RPE cells

The diphenyleneiodonium (DPI) is widely used as an inhibitor of flavoenzymes, particularly NADPH oxidase. In this study, we investigated the effect of DPI on the apoptosis of human RPE cells. DPI treatment in ARPE-19 cells evoked a dose- and time-dependent growth inhibition, and also induced DNA fragmentation and protein content of the proapoptotic factor Bax. In addition, DPI significantly induced the expression and phosphorylation of p53, which induces proapoptotic genes in response to DNA damage or irreparable cell cycle arrest. ROS have been implicated as a key factor in the activation of p53 by many chemotherapeutic drugs. Recent data on the regulation of intracellular ROS by DPI are controversial. Therefore, we analyzed whether DPI could contribute to the generation of intracellular ROS. Although there was increase in ROS level from cells treated for 24h with DPI, it was not detectable at early time points, required to induce p53 expression. And DPI-induced p53 expression was not affected by the ROS scavenger NAC. We conclude that DPI induces the expression of p53 by ROS-independent mechanism in ARPE-19 cells, and renders cells sensitive to drug-induced apoptosis by induction of p53 expression.     

NADPH Oxidase-Dependent Production of Reactive Oxygen Species Induces Endoplasmatic Reticulum Stress in Neutrophil-Like HL60 Cells

Reactive oxygen species (ROS) primarily produced via NADPH oxidase play an important role for killing microorganisms in neutrophils. In this study we examined if ROS production in Human promyelocytic leukemia cells (HL60) differentiated into neutrophil-like cells (dHL60) induces ER stress and activates the unfolded protein response (UPR). To cause ROS production cells were treated with PMA or by chronic hyperglycemia. Chronic hyperglycemia failed to induce ROS production and did not cause activation of the UPR in dHL60 cells. PMA, a pharmacologic NADPH oxidase activator, induced ER stress in dHL60 cells as monitored by IRE-1 and PERK pathway activation, and this was independent of calcium signaling. The NADPH oxidase inhibitor, DPI, abolished both ROS production and UPR activation. These results show that ROS produced by NADPH oxidase induces ER stress and suggests a close association between the redox state of the cell and the activation of the UPR in neutrophil-like HL60 cells.     

Reactive oxygen species are involved in eosinophil extracellular traps release and in airway inflammation in asthma

In asthma, there are high levels of inflammatory mediators, reactive oxygen species (ROS), and eosinophil extracellular traps (EETs) formation in airway. Here, we attempted to investigate the ROS involvement in EETs release and airway inflammation in OVA-challenged mice. Before the intranasal challenge with ovalbumin (OVA), animals were treated with two ROS inhibitors, N-acetylcysteine (NAC) or diphenyleneiodonium (DPI). We showed that NAC treatment reduced inflammatory cells in lung. DPI and NAC treatments reduced eosinophil peroxidase (EPO), goblet cells hyperplasia, proinflammatory cytokines, NFκB p65 immunocontent, and oxidative stress in lung. However, only the NAC treatment improved mitochondrial energy metabolism. Moreover, the treatments with DPI and NAC reduced EETs release in airway. This is the first study to show that ROS are needed for EETs formation in asthma. Based on our results, NAC and DPI treatments can be an interesting alternative for reducing airway inflammation, mitochondrial damage, and EETs release in asthma.     

Mechanism of neutrophil extracellular traps generation and their role in trophoblasts apoptosis in gestational diabetes mellitus

Gestational diabetes mellitus (GDM) is a metabolic syndrome occurring in pregnant women and increases the risk of placental dysplasia. Neutrophil extracellular traps (NETs) may play a critical role in placental dysplasia. NETosis (neutrophil cell death by NET release) depends on NADPH/ROS pathway. In view of the adiponectin which is widely believed to be reduced in GDM patients suppresses NADPH oxidase and ROS generation of neutrophil. We speculate that increased NET release is associated with hypoadiponectinemia. Trophoblast apoptosis is significantly increased in GDM patients, but it is not clear whether NETs promotes cell apoptosis. This study aims to reveal the mechanism of Neutrophil Extracellular Traps generation and their role in trophoblast apoptosis in Gestational Diabetes Mellitus. We investigated the generation of NETs by cell-free DNA (cf-DNA) quantification, live-cell imaging, and reactive oxygen species (ROS) measurement. ERK1/2 and p38 MAPK signalling pathway proteins were detected by western blotting. The Cell Counting Kit-8 (CCK-8) assay, flow cytometry, and western blotting were performed to explore the effects of NETs on trophoblast apoptosis. We found that adiponectin inhibited NET release by suppressing ROS production, and p38 MAPK and ERK1/2 proteins were involved in the process. Further, NETs promoted trophoblast apoptosis by activating the ROS-dependent mitochondrial pathway, which is mediated by ERK1/2 signalling. The current study demonstrated that hypoadiponectinemia is the cause of NETs formation and NETs promoting trophoblast apoptosis.     

Arrestin regulates MAPK activation and prevents NADPH oxidase-dependent death of cells expressing CXCR2

Activation of CXCR2 IL-8 receptor leads to activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and rapid receptor endocytosis. Co-immunoprecipitation and co-localization experiments showed that arrestin and CXCR2 form complexes with components of the ERK1/2 cascade following ligand stimulation. However, in contrast to the activation of the beta2-adrenergic receptor, arrestin was not necessary for ERK1/2 phosphorylation or receptor endocytosis. In contrast, beta-arrestin 1/2 double knockout cells showed greatly enhanced phosphorylation of ERK1/2, as well as phosphorylation of the stress kinases p38 and c-Jun N-terminal protein kinase. The stimulation of stress kinases in arrestin double knockout cells could be attenuated in the presence of diphenylene iodonium (DPI), an inhibitor of the NADPH oxidase, suggesting that reactive oxidant species (ROS) participated in mitogen-activated protein kinase (MAPK) activation. ROS could indeed be detected in IL-8-stimulated beta-arrestin 1/2 knockout cells, and cytoplasmic Rac was translocated to the membrane fraction, which is a prerequisite for oxidant formation. The oxidative burst induced cell death within 6 h of IL-8 stimulation of these cells, which could be prevented in the presence of DPI. These results indicate a novel function for arrestin, which is protection from an excessive oxidative burst, resulting from the sustained stimulation of G-protein-coupled receptors that cause Rac translocation.     

Carp neutrophilic granulocytes form extracellular traps via ROS-dependent and independent pathways

Neutrophil extracellular traps (NETs) have recently been described as an important innate defense mechanism that leads to immobilization and killing of invading pathogens. NETs have been identified in several species, but the mechanisms involved in NET formation and their role in infection have not been well determined yet. Here we show that upon in vitro stimulation with different immunostimulants of bacterial, fungal or viral origin, carp neutrophilic granulocytes rapidly release NET structures. We analyzed the composition of these structures and the kinetics of their formation by confocal microscopy, by quantifying the levels of extracellular DNA and the release of enzymes originating from neutrophilic granules: myeloperoxidase, neutrophil elastase and matrix metalloproteinase 9 (MMP-9). Profiles of NET release by carp neutrophils as well as their enzyme composition are stimulus- and time-dependent. This study moreover provides evidence for a stimulus-dependent selective requirement of reactive oxygen species in the process of NET formation. Collectively the results support an evolutionary conserved and strictly regulated mechanism of NET formation in teleost fish.     

Kaempferol blocks neutrophil extracellular traps formation and reduces tumour metastasis by inhibiting ROS‐PAD4 pathway

Kaempferol (kaem) is a dietary flavonoid found in a variety of fruits and vegetables. The inhibitory effects of kaem on primary tumour growth have been extensively investigated; however, its effects on tumour metastasis are largely unknown. In the present study, we found that kaem significantly suppresses both primary tumour growth and lung metastasis in mouse breast tumour model. Furthermore, decreased expression of citrullinated histone H3 (H3‐cit), a biomarker of neutrophil extracellular traps (NETs), had been founded in metastatic lung upon treated with kaem. The reduction of H3‐cit is not, however, due to the cytotoxicity of kaem on neutrophils since the frequency of CD11b⁺Ly6G⁺ neutrophils did not change in lung, tumour or blood in the presence of kaem. We then confirm the anti‐NETs effects of kaem in vitro by co‐culturing mouse neutrophils and kaem. Supplementing the neutrophils with GSK484, a potent NET inhibitor, totally abrogated the inhibitory effects of kaem on tumour metastasis while having little or no impact on primary tumour growth, indicating the specificity of kaem acting on NET formation and tumour metastasis. We also found that kaem suppressed ROS production in mouse bone‐marrow derived neutrophils. Supplementing with the ROS scavenger DPI abrogated kaem's effects on NET formation, suggesting the involvement of kaempferol in NADPH/ROS‐NETs signalling. Finally, we applied the kaem on NET‐deficient PAD4^‐/‐ mice and found decreased primary tumour volume and weight but similar lung metastatic tumour with kaempferol treatment. Therefore, our findings reveal a novel mechanism of kaem in breast cancer development by targeting NETs induced tumour metastasis.     

CD44/ERM/F‐actin complex mediates targeted nuclear degranulation and excessive neutrophil extracellular trap formation during sepsis

Neutrophils release neutrophil extracellular traps (NETs) to capture and kill pathogens, but excessive NET release can damage the surrounding tissues. Myeloperoxidase (MPO) and neutrophil elastase (NE) are thought to be important in promoting histone depolymerization and DNA breakage in the nucleus. However, the detailed path by which MPO and NE enter the nucleus is unknown. In the present study, we observed that delayed fusion of azurophilic granules with the nuclear membrane 15–20 min after extracellular degranulation in activated neutrophils. In a subsequent experiment, we further demonstrated that this fusion leads to MPO entry into the nucleus and promotes nuclear histone depolymerization and DNA breakage, a process called ‘targeted nuclear degranulation’. This process can be effectively inhibited by dexamethasone and accompanied by the continuous low levels of MPO in the nucleus after PMA stimulation. Meanwhile, we found that ‘targeted nuclear degranulation’ is dependent on the CD44 translocation and subsequent redistribution of CD44 / ERM (Ezrin/Radixin/Moesin) / F‐actin complexes, which guides the movement of azurophilic granules towards the nucleus. Application of ERM phosphorylation inhibitors and importin activity inhibitors significantly reduced the complexes formation and redistribution. Taken together, these findings indicate for the first time that delayed ‘targeted nuclear degranulation’ after neutrophil activation is a key mechanism of NET formation. CD44/ERM/F‐actin complex mediates this process, which providing targets with promising prospects for the precise regulation of NET formation.