溶酶体膜蛋白-2抗体诱导中性粒细胞胞外捕网形成的实验研究

目的研究溶酶体膜蛋白-2抗体(LAMP-2-antibody)诱导中性粒细胞胞外捕网形成(NETosis)的作用,为进一步探讨这种新型细胞死亡模式在系统性小血管炎中的作用及机制奠定基础。方法采用PolymorphprepTM进行密度梯度离心法分离健康志愿者外周静脉血中性粒细胞,抗LAMP-2抗体刺激中性粒细胞,以及通过NADPH氧化酶抑制剂二甲苯基碘(DPI)阻断活性氧簇的产生,电镜、免疫荧光观测胞外捕网的形成,激光共聚焦检测自身抗原LAMP-2、PR3、MPO的表达。结果 LAMP-2抗体促进中性粒细胞胞外捕网NETs形成,自身抗原MPO、PR3及LAMP-2包含在NETs内,抑制NADPH氧化酶的活性可以阻断NETs形成。结论 LAMP-2抗体可能通过诱导中性粒细胞异常死亡,释放内源性危险信号,激活自身免疫反应等来维持血管炎的恶性持续状态。     

中性粒细胞胞外诱捕网形成的分子机制研究进展

中性粒细胞作为固有免疫的一线细胞,其死亡形式-中性粒细胞胞外诱捕网（NETs）过度形成过程中因释放大量胞内成分而参与包括自身免疫性疾病在内的多种疾病发生。自身免疫性疾病以自身抗原暴露、免疫耐受缺失为特征,且目前治疗仍以改善症状为主,如能抑制自身抗原产生将为其根治提供新的可能。本文主要对参与NETs形成的分子进行综述,包括ROS、PAD-4、自噬以及相关信号通路,以期促进NETs相关疾病治疗策略的开发。     

ELR+ CXC趋化因子诱导NETs生成的作用机制

目的探讨ELR~+ CXC趋化因子诱导中性粒细胞胞外诱捕网(neutrophil extracellular traps,NETs)生成的作用及机制。方法分离人外周血中性粒细胞,通过荧光染色和总MPO定量检测比较ELR~+ CXC趋化因子、ELR-CXC趋化因子和非趋化性细胞因子对NETs的诱导作用。以CXCL8为代表性ELR~+ CXC趋化因子,检测其诱导NETs的时效、量效关系,明确参与NETs生成的酶类成分,胞膜受体以及胞内信号分子机制。检测CXCL8对中性粒细胞ROS的上调效应及其在介导NETs生成中的作用。结果仅有ELR~+ CXC趋化因子可刺激NETs释放(P0.05或0.01)。其代表性因子CXCL8诱导NETs具有时间和剂量依赖性。抑制MPO、NE和PAD4活化,或者抑制CXCR1受体内化以及胞内ERK和p38的磷酸化,均可显著下调CXCL8诱导的NETs生成(P0.01)。CXCL8还通过上调NOX2并增加胞内ROS水平介导NETs生成。结论 ELR~+ CXC趋化因子可诱导NETs生成,该作用与其结合CXCR1,激活ERK和p38,诱导NOX2活化和上调ROS生成有关。     

肺炎链球菌诱导中性粒细胞胞外捕获网形成

目的初步探究肺炎链球菌诱导中性粒细胞胞外捕获网(NETs)形成的作用及调节机制,为进一步研究奠定基础。方法采用免疫磁珠法分选C57BL/6小鼠骨髓中性粒细胞,肺炎链球菌刺激中性粒细胞,分别采用脱氧核糖核酸酶(DNase)降解NETs和NADPH氧化酶抑制剂二苯基氯化碘盐(DPI)预处理中性粒细胞阻断活性氧簇(ROS)生成,免疫荧光染色观察NETs结构,检测游离DNA定量观察NETs的生成。结果肺炎链球菌刺激中性粒细胞释放NETs,DNase降解NETs结构,阻断ROS生成后NETs形成受抑。结论肺炎链球菌能够诱导NETs形成,DNase直接降解NETs或DPI抑制NETs形成可以成为调节NETs生成的有效途径。     

白蛋白对脂多糖诱导中性粒细胞胞外诱捕网生成的抑制作用

目的探讨白蛋白对脂多糖(LPS)诱导人外周血中性粒细胞胞外诱捕网(neutrophil extracellular traps,NETs)生成的抑制作用及机制。方法分离人外周血中性粒细胞,分别培养于无白蛋白和含生理浓度白蛋白的培养体系中,加入LPS刺激,同时使用钙离子(Ca²⁺)以及自噬调节剂干预,采用sytox green染色法检测NETs,采用Fluo-4 AM染色法检测胞内Ca²⁺水平,免疫荧光法检测NETs相关蛋白髓过氧化物酶(MPO)、弹性蛋白酶(NE)和瓜氨酸化组蛋白H3(cit-H3)以及自噬体相关蛋白LC3B的表达。结果 LPS可诱导中性粒细胞释放NETs,白蛋白可抑制NETs生成;白蛋白可降低LPS刺激下中性粒细胞胞内Ca²⁺浓度和自噬水平。Ca²⁺载体可增强中性粒细胞自噬水平并促进LPS诱导的NETs生成,Ca²⁺螯合剂可降低自噬水平并抑制NETs生成。自噬激动剂可显著增强NETs,自噬抑制剂则显著抑制NETs。结论白蛋白对LPS诱导中性粒细胞胞外诱捕网(N...     

中性粒细胞胞外诱捕网形成机制的研究进展

中性粒细胞主要通过吞噬、脱颗粒和产生活性氧(ROS)等方式行使功能,后来发现中性粒细胞还可以通过形成中性粒细胞胞外诱捕网(NET)参与固有免疫反应, NET的形成过程被称为中性粒细胞胞外诱捕网凋零(NETosis),属于细胞程序性死亡方式。中性粒细胞激活后,经过产生ROS、弹性蛋白酶(NE)和髓过氧化物酶(MPO)转运到细胞核,引起组蛋白修饰、染色质解凝、细胞膜破裂,形成NET,被称为依赖烟酰胺腺嘌呤二核苷酸磷酸(NADPH)的NETosis;不依赖NADPH的NETosis则需要肽基精氨酸脱氨酶4(PAD4)的参与;此外,还有NET快速形成过程。     

中性粒细胞胞外诱捕网与相关疾病的研究进展

中性粒细胞是机体防御系统的第一道防线,当机体遭受病原微生物的入侵,中性粒细胞即发挥强大的作用.研究发现,活化的中性粒细胞可以释放出非浓缩染色质形成网状支架,对病原体进行包围及限制,这一过程称为中性粒细胞胞外陷阱(neutrophil extracellular traps,NETs)形成(NETosis).NETs是由DNA骨架、组蛋白、颗粒成分以及胞浆蛋白组成的网状物,其能够网罗、杀伤病原体从而参与机体自身免疫应答,维护机体健康.然而,中性粒细胞胞外诱捕网的产生是一把双刃剑,近年来有研究表明,它可以引发代谢性疾病,如糖尿病(diabetes mellitus,DM),引起心血管疾病动脉粥样硬化(atherosclerosis,AS),参与自身免疫性疾病,如系统性红斑狼疮(system lupus erythematosus systemic lupus erythematosus,SLE)、类风湿性关节炎(rheumatoid arthritis,RA)的产生,还可以使自身凝血异常,刺激血栓形成并为其提供支架,因而研究NETs及其与疾病之间的关系具有重要意义.     

活性氧在中性粒细胞胞外诱捕网形成中的作用

中性粒细胞胞外诱捕网(NETs)是胞外的一种DNA网状结构,其可网罗、杀伤病原体从而参与机体固有免疫应答.然而在某些情况下NETs会引发一系列自身免疫性疾病.研究表明,在NETs形成过程中活性氧(ROS)起到至关重要的作用.阐述活性氧与NETs形成的相互作用,进一步讨论了尚未完全明确的活性氧与NETs的关系,包括还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)氧化酶产生的ROS的作用,依赖NADPH氧化酶产生NETs的分子机制,以及非NADPH氧化酶产生的ROS的作用,从多方面分析ROS与NETs形成的关系.     

G 试验阳性血清通过干扰 Dectin-1的内在化表达抑制 ROS 依赖性杀伤白念珠菌北大核心CSCD

目的：探讨G试验阳性血清中游离β-1,3-D-葡聚糖干扰人嗜中性粒细胞Dectin-1所介导的氧依赖性杀伤白念珠菌的机制。方法激光共聚焦显微镜检测经β-1,3-D-葡聚糖酶预处理前后的白念珠菌孢子作用下,人嗜中性粒细胞内Dectin-1表达分布的变化及与活性氧簇（ reactive oxy-gen species, ROS）的共定位关系;进一步分析Dectin-1的阻断对白念珠菌诱导下胞内ROS生成的影响;流式细胞术检测G试验阳性血清中游离的β-1,3-D-葡聚糖对白念珠菌诱导下人嗜中性粒细胞内Dectin-1和ROS表达水平的影响。结果在白念珠菌作用下,人嗜中性粒细胞内诱导性表达的Dec-tin-1主要被募集到内吞的孢子表面,且与ROS的产生具有一定的共定位关系。然而,经β-1,3-D-葡聚糖酶预处理后,胞内则未见Dectin-1的明显表达;当Dectin-1被其功能性抗体（5μg/ml）阻断后,白念珠菌诱导胞内ROS的表达与阻断前相比显著降低（LSD-t检验,P＜0．01）;经G试验阳性血清预处理的人嗜中性粒细胞中,白念珠菌诱导下胞内Dectin-1和ROS的表达水平均显著低于单一的白念珠菌作用组（LSD-t检验, P＜0．01）;线性回归分析提示G试验阳性血清中游离β-1,3-D-葡聚糖浓度分别与Dectin-1及ROS表达水平的抑制部分呈显著的剂量依存关系（ Dectin-1,R2＝0．702,P＜0．01;ROS,R2＝0．588,P＜0．01）。结论 G试验阳性血清中的游离β-1,3-D-葡聚糖可通过干扰人嗜中性粒细胞Dectin-1内在化表达的方式抑制ROS依赖性杀伤内吞的白念珠菌孢子。     

ANCA诱导中性粒细胞释放正五聚素蛋白3*

 目的：研究正五聚素蛋白3（PTX3）在中性粒细胞的表达及释放情况,为进一步探讨PTX3在抗中性粒细胞胞浆抗体（ANCA）相关性小血管炎（AAV）中的作用及机制奠定基础。方法：采用PolymorphprepTM进行密度梯度离心分离健康志愿者和AAV患者外周静脉血中性粒细胞,通过健康人血清、AAV患者血清、TNF-α和溶酶体膜蛋白2（LAMP-2）抗体分别刺激健康人外周血中性粒细胞,ELISA检测培养上清PTX3水平,免疫荧光观察中性粒细胞胞外捕网(NETs)的形成,激光共聚焦显微镜检测可溶性识别受体PTX3 的表达。结果：LAMP-2抗体及AAV患者血清均能促进中性粒细胞PTX3的释放。与正常中性粒细胞对照组相比,LAMP-2抗体刺激组中性粒细胞上清PTX3水平明显升高（P＜0.01）; AAV患者血清刺激组中性粒细胞中PTX3表达的荧光强度显著高于用健康人血清刺激的对照组（P＜0.01）,进一步通过激光共聚焦显微镜进行形态学观察,发现PTX3由中性粒细胞通过NETs形成释放。结论：PTX3可由ANCA诱导的NETs释放至胞外,参与血管炎的病理生理过程。     

Neutrophil extracellular traps generation and degradation in patients with granulomatosis with polyangiitis and systemic lupus erythematosus

Neutrophils are one of the first cells to arrive at the site of infection, where they apply several strategies to kill pathogens: degranulation, respiratory burst, phagocytosis, and release of neutrophil extracellular traps (NETs). Recent discoveries try to connect NETs formation with autoimmune diseases, like systemic lupus erythematosus (SLE) or granulomatosis with polyangiitis (GPA) and place them among one of the factors responsible for disease pathogenesis. The aim of the study was to assess the NETotic capabilities of neutrophils obtained from freshly diagnosed autoimmune patients versus healthy controls. Further investigation involved assessing NETs production among treated patients. In the latter step, NETs degradation potency of collected sera from non-treated patients was checked. Lastly, the polymorphisms of the DNASE I gene among tested subjects were checked. NETs formation was measured in a neutrophil culture by fluorometry, while degradation assessment was performed with patients’ sera and extracellular source of DNA. Additionally, Sanger sequencing was used to check potential SNP mutations between patients. About 121 subjects were enrolled into this study, 54 of them with a diagnosed autoimmune disorder. Neutrophils stimulated with NETosis inducers were able to release NETs in all cases. We have found that disease affected patients produce NETs more rapidly and in larger quantities than control groups, with up to 82.5% more released. Most importantly, we showed a difference between the diseases themselves. NETs release was 68.5% higher in GPA samples when compared to SLE ones while stimulated with Calcium Ionophore. Serum nucleases were less effective at degrading NETs in both autoimmune diseases, with a reduction in degradation of 20.9% observed for GPA and 18.2% for SLE when compared with the controls. Potential therapies targeting neutrophils and NETs should be specifically tailored to the type of the disease. Since there are significant differences between NETs release and disease type, a standard neutrophil targeted therapy could prevent over-generation of traps in some cases, while in others it would deplete the cells, leaving the immune system unresponsive to primary infections.     

Neutrophils activate plasmacytoid dendritic cells by releasing self-DNA-peptide complexes in systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a severe and incurable autoimmune disease characterized by chronic activation of plasmacytoid dendritic cells (pDCs) and production of autoantibodies against nuclear self-antigens by hyperreactive B cells. Neutrophils are also implicated in disease pathogenesis; however, the mechanisms involved are unknown. Here, we identified in the sera of SLE patients immunogenic complexes composed of neutrophil-derived antimicrobial peptides and self-DNA. These complexes were produced by activated neutrophils in the form of web-like structures known as neutrophil extracellular traps (NETs) and efficiently triggered innate pDC activation via Toll-like receptor 9 (TLR9). SLE patients were found to develop autoantibodies to both the self-DNA and antimicrobial peptides in NETs, indicating that these complexes could also serve as autoantigens to trigger B cell activation. Circulating neutrophils from SLE patients released more NETs than those from healthy donors; this was further stimulated by the antimicrobial autoantibodies, suggesting a mechanism for the chronic release of immunogenic complexes in SLE. Our data establish a link between neutrophils, pDC activation, and autoimmunity in SLE, providing new potential targets for the treatment of this devastating disease.     

Acetylated histones contribute to the immunostimulatory potential of neutrophil extracellular traps in systemic lupus erythematosus

In addition to disturbed apoptosis and insufficient clearance of apoptotic cells, there is recent evidence for a role of neutrophils in the aetiopathogenesis of systemic lupus erythematosus (SLE). In response to various stimuli, neutrophils can rapidly release DNA fibres decorated with citrullinated histones and anti-microbial peptides. These structures are referred to as neutrophil extracellular traps (NETs). In addition to apoptotic cell-derived microparticles, these NETs may comprise a further source of autoantigens, able to drive the autoimmune response in SLE. Our group recently identified specific histone modifications occurring during apoptosis that play an important role in the autoimmune response in SLE. In the current study, we evaluated the presence and immunostimulatory potential of these previously identified histone modifications in NETs. Compared to NETs from healthy donors, the histones present in NETs formed by SLE-derived neutrophils contain increased amounts of acetylated and methylated residues, which we previously observed to be associated with apoptosis and SLE. Treatment of neutrophils with histone deacetylase (HDAC) inhibitor Trichostatin A (TSA), prior to induction of NETosis, induced NETs containing hyperacetylated histones, endowed with an increased capacity to activate macrophages. This implies that specific histone modifications, in particular acetylation, might enhance the immunostimulatory potential of NETs in SLE.     

Neutrophil extracellular traps and neutrophil-derived mediators as possible biomarkers in bronchial asthma

Neutrophils (PMNs) contain and release a powerful arsenal of mediators, including several granular enzymes, reactive oxygen species (ROS) and neutrophil extracellular traps (NETs). Although airway neutrophilia is associated with severity, poor response to glucocorticoids and exacerbations, the pathophysiological role of neutrophils in asthma remains poorly understood. Twenty-four patients with asthma and 22 healthy controls (HCs) were prospectively recruited. Highly purified peripheral blood neutrophils (> 99%) were evaluated for ROS production and activation status upon stimulation with lipopolysaccharide (LPS), N-formylmethionyl-leucyl-phenylalanine (fMLP) and phorbol 12-myristate 13-acetate (PMA). Plasma levels of myeloperoxidase (MPO), CXCL8, matrix metalloproteinase-9 (MMP-9), granulocyte–monocyte colony-stimulating factor (GM-CSF) and vascular endothelial growth factor (VEGF-A) were measured by ELISA. Plasma concentrations of citrullinated histone H3 (CitH3) and circulating free DNA (dsDNA) were evaluated as NET biomarkers. Activated PMNs from asthmatics displayed reduced ROS production and activation status compared to HCs. Plasma levels of MPO, MMP-9 and CXCL8 were increased in asthmatics compared to HCs. CitH3 and dsDNA plasma levels were increased in asthmatics compared to controls and the CitH3 concentrations were inversely correlated to the % decrease in FEV₁/FVC in asthmatics. These findings indicate that neutrophils and their mediators could have an active role in asthma pathophysiology.

     

Procoagulant role of neutrophil extracellular traps in patients with gastric cancer

Background: Patients with gastric cancer (GC) commonly exhibit a hypercoagulable state that results in significant morbidity and mortality. Recent studies have shown that neutrophil extracellular traps (NETs) trigger coagulation through an intrinsic pathway and contribute to thrombus initiation and progression. In this study, we aimed to determine the procoagulant activity (PCA) of NETs in patients with GC. Methods: NET formation and their PCAs were assessed in 48 patients with GC and 36 healthy controls using immunofluorescence microscopy of neutrophil markers and extracellular DNA as well as a modified capture ELISA technique, and thrombin-antithrombin complex and clot (fibrin) spectroscopic detection, respectively. Results: Here we showed that neutrophils isolated from patients with GC displayed significantly enhanced NET formation compared with those from healthy controls; furthermore, plasma or platelets obtained from patients with GC induced control neutrophils to release NETs. In addition, NETs released by GC neutrophils significantly increased the potency of control plasma to generate thrombin and fibrin. Notably, these procoagulant effects were dramatically attenuated by application of DNase I. We further found that spontaneous NET formation in patients with GC was significantly higher than that in controls, increased with tumor- node-metastasis stage elevation, and positively correlated with thrombin-antithrombin complex levels and D-dimers. Additionally, the effect of DNase I on cell-free plasma generation of fibrin was dependent on the concentration of NET formation. Conclusion: These results suggest that GC creates a systemic environment that primes neutrophils to release procoagulant NETs. Thus, targeting NETs might improve the coagulopathy of patients with GC.     

Inhibition of neutrophil-mediated production of reactive oxygen species (ROS) by endothelial cells is not impaired in anti-neutrophil cytoplasmic autoantibodies (ANCA)-associated vasculitis patients

Leucocyte transendothelial migration is strictly regulated to prevent undesired inflammation and collateral damage of endothelial cells by activated neutrophils/monocytes. We hypothesized that in anti‐neutrophil cytoplasmic autoantibodies (ANCA)‐associated vasculitis (AAV) patients' dysregulation of this process might underlie vascular inflammation. Peripheral blood mononuclear cells (PBMC) and neutrophils from AAV patients (n = 12) and healthy controls (HC, n = 12) were isolated. The influence of human umbilical vein endothelial cells (HUVEC) on neutrophil/monocytes function was tested by N‐formyl‐methionyl‐leucyl‐phenyl‐alanine (fMLP)‐ and phorbol 12‐myristate 13‐acetate (PMA)‐mediated ROS production, degranulation and interleukin (IL)‐8 production. In addition, the ability of lipopolysaccharide (LPS)‐stimulated PBMC to produce tumour necrosis factor (TNF)‐α in the presence or absence of HUVEC was tested. HUVEC inhibited ROS production dose‐dependently by fMLP‐stimulated neutrophils but did not influence degranulation. No differences between neutrophils from HC and AAV were found. However, in only one active patient was degranulation inhibited significantly by HUVEC only before cyclophosphamide treatment, but not 6 weeks later. Co‐cultures of HUVEC with LPS‐stimulated neutrophils/monocytes increased IL‐8 production while TNF‐α production was inhibited significantly. There was no apparent difference between AAV patients and HC in this respect. Our findings demonstrate that HUVEC are able to inhibit ROS and modulate cytokine production upon stimulation of neutrophils or monocytes. Our data do not support the hypothesis that endothelial cells inhibit ROS production of neutrophils from AAV patients inadequately. Impaired neutrophil degranulation may exist in active patients, but this finding needs to be confirmed.     

Reactive Oxygen Species in Peripheral Blood and Sputum Neutrophils During Bacterial and Nonbacterial Acute Exacerbation of Chronic Obstructive Pulmonary Disease

Chronic airway inflammation can be mediated by an enhanced neutrophil oxidative burst. However, the role of bacteria in the pathogenesis of chronic obstructive pulmonary disease (COPD) exacerbations is highly controversial. The aim of this study was to evaluate the production of reactive oxygen species (ROS) in peripheral blood and sputum neutrophils during bacterial and nonbacterial acute exacerbations of COPD (AECOPD). A total of 40 patients with AECOPD, 10 healthy nonsmokers, and 10 “healthy” smokers were enrolled into the study. Peripheral blood and sputum samples were obtained during exacerbation and after recovery. Neutrophils were isolated by high-density gradient centrifugation and magnetic separation. ROS production by neutrophils was investigated after stimulation with phorbol-myristate-acetate and Staphylococcus aureus bacteria. ROS production by neutrophils was assessed as the mean fluorescent intensity using a flow cytometer. IL-8 levels in serum and induced sputum were determinant by ELISA. Spontaneous ROS production was significantly higher in neutrophils from the patients with bacterial AECOPD as compared with nonbacterial AECOPD and stable COPD (P <0.05). ROS production stimulated with PMA and with Staphylococcus aureus was significantly higher in neutrophils isolated from the patients with bacterial AECOPD as compared with nonbacterial and stable COPD (P <0.05). The serum and induced sputum IL-8 levels were significantly increased in the patients with bacterial AECOPD than nonbacterial AECOPD, stable COPS, and “healthy” smokers and nonsmokers (P <0.05) and higher in the induced sputum as the compared with serum in all studied groups (P <0.05). Enlarge CRP level was documented during AECOPD than in all other groups (P <0.05). A markedly increased ROS production in sputum neutrophils during bacterial AECOPD shows an inflammatory response reflecting enhanced local inflammation, which can be mediated by bacterial colonization.     

Carrageenan‐induced inflammation promotes ROS generation and neutrophil extracellular trap formation in a mouse model of peritonitis

Neutrophil extracellular traps (NETs) are a combination of DNA fibers and granular proteins, such as neutrophil elastase (NE). NETs are released in the extracellular space in response to different stimuli. Carrageenan is a sulfated polysaccharide extracted from Chondrus crispus, a marine algae, used for decades in research for its potential to induce inflammation in different animal models. In this study, we show for the first time that carrageenan injection can induce NET release in a mouse model of acute peritonitis. Carrageenan induced NET release by viable neutrophils with NE and myeloperoxidase (MPO) expressed on DNA fibers. Furthermore, although this polysaccharide was able to stimulate reactive oxygen species (ROS) generation by peritoneal neutrophils, NADPH oxidase derived ROS were dispensable for NET formation by carrageenan. In conclusion, our results show that carrageenan‐induced inflammation in the peritoneum of mice can induce NET formation in an ROS‐independent manner. These results may add important information to the field of inflammation and potentially lead to novel anti‐inflammatory agents targeting the production of NETs.     

PMA and crystal‐induced neutrophil extracellular trap formation involves RIPK1‐RIPK3‐MLKL signaling

Neutrophil extracellular trap (NET) formation contributes to gout, autoimmune vasculitis, thrombosis, and atherosclerosis. The outside‐in signaling pathway triggering NET formation is unknown. Here, we show that the receptor‐interacting protein kinase (RIPK)‐1‐stabilizers necrostatin‐1 or necrostatin‐1s and the mixed lineage kinase domain‐like (MLKL)‐inhibitor necrosulfonamide prevent monosodium urate (MSU) crystal‐ or PMA‐induced NET formation in human and mouse neutrophils. These compounds do not affect PMA‐ or urate crystal‐induced production of ROS. Moreover, neutrophils of chronic granulomatous disease patients are shown to lack PMA‐induced MLKL phosphorylation. Genetic deficiency of RIPK3 in mice prevents MSU crystal‐induced NET formation in vitro and in vivo. Thus, neutrophil death and NET formation may involve the signaling pathway defining necroptosis downstream of ROS production. These data imply that RIPK1, RIPK3, and MLKL could represent molecular targets in gout or other crystallopathies.