Dermatophagoides farinae induces conjunctival epithelial cell damage to promote neutrophil migration and neutrophil extracellular traps formation / 中国血吸虫病防治杂志

OBJECTIVE@#To investigate the mechanisms underlying allergic conjunctivitis caused by conjunctival epithelial cell damage, neutrophil migration and neutrophil extracellular traps (NETs) formation induced by crude extracts of Dermatophagoides farinae mite (CDM).@*METHODS@#Human conjunctival epithelial cells were stimulated with 500, 1 000, 2 000, 4 000 ng/mL, and the expression levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ) and IL-8 were detected using quantitative real-time PCR (qPCR) assay and enzyme-linked immunosorbent assay (ELISA). The culture supernatant of human conjunctival epithelial cells was collected and co-cultured with neutrophils. Neutrophil migration was measured using Transwell migration assay, and the expression of NETs markers myeloperoxidase (MPO) and citrullinated histone H3 (CitH3) was quantified using immunofluorescence staining. Neutrophils were stimulated with phorbol 12-myristate 13-acetate (PMA), and then NETs were collected for treatment of human conjunctival epithelial cells. Cell apoptosis was detected using flow cytometry, and the levels of IL-6, TNF-α, IFN-γ and IL-8 were measured in the cell culture supernatant using ELISA.@*RESULTS@#Treatment with CDM at concentrations of 2 000 ng/mL and 4 000 ng/mL up-regulated IL-6, TNF-α, IFN-γ and IL-8 expression in human conjunctival epithelial cells. Following treatment with CDM at concentrations of 2 000 ng/mL and 4 000 ng/mL, the culture supernatant of human conjunctival epithelial cells promoted neutrophil migration and induced increases in the staining intensity of MPO and CitH3. In addition, increased NETs triggered the apoptosis of human conjunctival epithelial cells and IL-6, TNF-α, IFN-γ and IL-8 secretion in the culture supernatant of human conjunctival epithelial cells.@*CONCLUSIONS@#CDM induces human conjunctival epithelial cell damages, thereby promoting neutrophil migration and NETs formation, while the release of NETs further aggravates human conjunctival epithelial cell damages.

Levels of neutrophil extracellular traps in neonates with acute respiratory distress syndrome / 中国当代儿科杂志

OBJECTIVES@#To study the changes in cell free-DNA (cf-DNA), a marker of neutrophil extracellular traps (NETs), in neonates with acute respiratory distress syndrome (ARDS), and to evaluate its relationship with the severity and early diagnosis of ARDS.@*METHODS@#The neonates diagnosed with ARDS in the Affiliated Hospital of Jiangsu University from January 2021 to June 2022 were enrolled in the prospective study. The neonates were divided into mild, moderate, and severe ARDS groups based on the oxygen index (OI) (4≤OI<8, 8≤OI<16, and OI≥16, respectively). The control group was selected from jaundice neonates who were observed in the neonatal department of the hospital during the same period, and they had no pathological factors causing neonatal jaundice. Peripheral blood samples were collected on day 1, day 3, and day 7 after admission for the ARDS group, and on the day of admission for the control group. Serum cf-DNA levels were measured using a fluorescence enzyme-linked immunosorbent assay. Serum interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) levels were measured using enzyme-linked immunosorbent assay. A Pearson correlation analysis was used to evaluate the correlation of serum cf-DNA levels with serum IL-6 and TNF-α levels.@*RESULTS@#A total of 50 neonates were enrolled in the ARDS group, including 15 neonates with mild ARDS, 25 with moderate ARDS, and 10 with severe ARDS. Twenty-five neonates were enrolled in the control group. Compared with the control group, the serum levels of cf-DNA, IL-6, and TNF-α in all ARDS groups were significantly increased (P<0.05). Compared with the mild ARDS group, the serum levels of cf-DNA, IL-6, and TNF-α in the moderate and severe ARDS groups were significantly increased (P<0.05), and the increase was more significant in the severe ARDS group (P<0.05). The serum levels of cf-DNA, IL-6, and TNF-α in all ARDS groups were significantly increased on day 3 after admission and significantly decreased on day 7 after admission compared with those on day 1 after admission (P<0.05). The Pearson correlation analysis showed that there was a positive correlation between serum cf-DNA levels and IL-6 levels as well as TNF-α levels in 50 neonates with ARDS (P<0.05).@*CONCLUSIONS@#There is an excessive expression of NETs in neonates with ARDS, and dynamic monitoring of serum cf-DNA levels has certain clinical value in evaluating the severity and early diagnosis of ARDS in neonates.

Neutrophil extracellular traps activates focal adhesion kinase by upregulating MMP9 expression to promote proliferation and migration of mouse colorectal cancer cells / 细胞与分子免疫学杂志

Objective To investigate how the neutrophil extracellular traps (NETs) affect the proliferation and migration of mouse MC38 colorectal cancer cells and its mechanism. Methods Spleen neutrophils were extracted in mouse, followed by collection of NETs after ionomycin stimulation in vitro. The proliferation of MC38 cell was detected by CCK-8 assay, and migration ability were detected by TranswellTM and cell scratch assay, after co-incubation with MC38 cells. The mRNA expression of cellular matrix metalloproteinase 2 (MMP2) and MMP9 were detected by real-time fluorescence quantitative PCR, and the expression of MMP2, MMP9 and focal adhesion kinase (FAK), phosphorylated FAK protein were detected by Western blot. After silencing MMP9 using small interfering RNA (siRNA), the effect of NETs on the proliferation and migration ability of MC38 cells and the altered expression of related molecules were examined by previous approach. Results NETs promoted the proliferation and migration of MC38 cells and up-regulated the MMP9 expression and FAK phosphorylation. Silencing MMP9 inhibited the promotion of MC38 proliferation and migration by NETs and suppressed FAK phosphorylation. Conclusion NETs up-regulates MMP9 expression in MC38 cells, activates FAK signaling pathway and promotes tumor cell proliferation and migration.

Research progress on the relationship between low-density neutrophils and infectious diseases / 细胞与分子免疫学杂志

Neutrophils play an important role in infectious diseases by clearing pathogens in the early stages of the disease and damaging the surrounding tissues along with the disease progress. Low-density neutrophils (LDNs) are a crucial and distinct subpopulation of neutrophils. They are a mixture of activated and degranulated normal mature neutrophils and a considerable number of immature neutrophils prematurely released from the bone marrow. Additionally, they may be involved in the occurrence and development of diseases through the changes in phagocytosis, the generation of reactive oxygen species (ROS), the enhancement of the ability to produce neutrophils extracellular traps and immunosuppression. We summarizes the role of LDNs in the pathogenesis and their correlation with the severity of infectious diseases such as COVID-19, severe fever with thrombocytopenia syndrome (SFTS), AIDS, and tuberculosis.

Increased autophagy of peripheral blood neutrophils and neutrophils extracellular traps formation in systemic lupus erythematosus / 细胞与分子免疫学杂志

Objective To explore the role of autophagy, apoptosis of neutrophils and neutrophils extracellular traps (NET) formation in systemic lupus erythematosus (SLE). Methods Thirty-six patients with SLE were recruited as research subjects, and 32 healthy controls matched accordingly were enrolled as control subjects. The expression levels of microtubule associated protein 1 light chain 3B (LC3B), autophagy-related gene5(ATG5), P62, B-cell lymphoma 2(Bcl2), Bcl2-related X protein (BAX) in neutrophils were detected by Western blot analysis. Flow cytometry was employed to analyze the expression of LC3B on neutrophils. The expression level of myeloperoxidase(MPO) in plasma was estimated by ELISA. Furthermore, neutrophils were cultured in vitro and stimulated by 100 nmol/L rapamycin and 10 μg/mL lipopolysaccharide (LPS) for 6 hours, respectively. And then, the expression levels of LC3B, ATG5, P62, Bcl2 and BAX in neutrophils were detected by Western blot analysis. The level of MPO in culture supernatant was detected by ELISA. The change of fluorescence intensity of NET in culture supernatant was assayed by SytoxTM Green staining combined with fluorescence spectrophotometry. Results Compared with healthy controls, the levels of autophagy and apoptosis of neutrophils and NET formation in SLE patients were increased. The level of apoptosis and NET formation was positively associated with neutrophil autophagy. The level of autophagy showed an increase but had no effect on apoptosis and NET formation for neutrophil stimulated by rapamycin. The levels of autophagy and NET formation also increased with no significant effect on apoptosis for neutrophil induced by LPS. Conclusion The autophagy, apoptosis and NET formation of neutrophils increase in SLE patients. The activation of autophagy and NET in neutrophils possibly result from the inflammatory internal environment in SLE patients.

Effects of neutrophil extracellular traps during human respiratory syncytial virus infection in vitro / Efeitos das redes extracelulares dos neutrófilos durante a infecção in vitro pelo vírus sincicial respiratório

Abstract The human respiratory syncytial virus (hRSV) is the most common cause of severe lower respiratory tract diseases in young children worldwide, leading to a high number of hospitalizations and significant expenditures for health systems. Neutrophils are massively recruited to the lung tissue of patients with acute respiratory diseases. At the infection site, they release neutrophil extracellular traps (NETs) that can capture and/or inactivate different types of microorganisms, including viruses. Evidence has shown that the accumulation of NETs results in direct cytotoxic effects on endothelial and epithelial cells. Neutrophils stimulated by the hRSV-F protein generate NETs that are able to capture hRSV particles, thus reducing their transmission. However, the massive production of NETs obstructs the airways and increases disease severity. Therefore, further knowledge about the effects of NETs during hRSV infections is essential for the development of new specific and effective treatments. This study evaluated the effects of NETs on the previous or posterior contact with hRSV-infected Hep-2 cells. Hep-2 cells were infected with different hRSV multiplicity of infection (MOI 0.5 or 1.0), either before or after incubation with NETs (0.5-16 μg/mL). Infected and untreated cells showed decreased cellular viability and intense staining with trypan blue, which was accompanied by the formation of many large syncytia. Previous contact between NETs and cells did not result in a protective effect. Cells in monolayers showed a reduced number and area of syncytia, but cell death was similar in infected and non-treated cells. The addition of NETs to infected tissues maintained a similar virus-induced cell death rate and an increased syncytial area, indicating cytotoxic and deleterious damages. Our results corroborate previously reported findings that NETs contribute to the immunopathology developed by patients infected with hRSV.

Resumo O vírus sincicial respiratório humano (hRSV) é a causa mais comum de doenças graves do trato respiratório inferior em crianças pequenas em todo o mundo, resultando em grande número de hospitalizações e gastos significativos para os sistemas de saúde. Neutrófilos são recrutados em massa para o tecido pulmonar de pacientes com doenças respiratórias agudas. No local da infecção, eles liberam armadilhas extracelulares de neutrófilos (NETs) que podem capturar e/ou inativar diferentes tipos de microrganismos, incluindo vírus. Evidências demonstraram que o acúmulo de NETs resulta em efeitos citotóxicos diretos nas células endoteliais e epiteliais. Os neutrófilos estimulados pela proteína F do vírus sincicial respiratório (hRSV-F) geram NETs que são capazes de capturar partículas virais, reduzindo assim sua transmissão. No entanto, a produção maciça de NETs obstrui as vias aéreas e aumenta a gravidade da doença. Assim, um maior conhecimento sobre os efeitos das NETs durante as infecções por hRSV é essencial para o desenvolvimento de novos tratamentos específicos e eficazes. Este estudo avaliou os efeitos das NETs no contato prévio ou posterior à infecção de células Hep-2 com hRSV. As células Hep-2 foram infectadas com diferentes quantidades de hRSV (multiplicidade de infecção ou MOI 0,5 ou 1,0), antes ou após a incubação com NETs (0,5-16 μg/mL). Células infectadas e não tratadas mostraram redução da viabilidade celular e intensa coloração com azul de tripano, que foi acompanhada pela formação de sincícios numerosos e grandes. O contato prévio entre as NETs e as células não resultou em efeito protetor. As células em monocamadas mostraram um número e área de sincícios reduzidos, mas a morte celular foi semelhante àquela apresentada por células infectadas e não tratadas. A adição de NETs aos tecidos infectados manteve taxa de morte celular e formação de sincícios semelhantes àqueles induzidos pelo vírus em células não tratadas, indicando danos citotóxicos e deletérios. Nossos resultados corroboram achados relatados anteriormente de que as NETs contribuem para a imunopatologia desenvolvida por pacientes infectados com hRSV.

Periodontite associada à artrite reumatoide: o papel das redes extracelulares de neutrófilos (NETs) / Periodontitis associated with rheumatoid arthritis: the role of neutrophil extracellular traps (NETs)

Introdução: As redes extracelulares de neutrófilos (NETs) são apontadas como um dos mecanismos relevantes na patogênese da periodontite e artrite reumatoide (AR). No entanto, permanece pouco compreendido a participação das NETs como mecanismo de ligação entre as duas doenças. Objetivos: 1) Investigar a concentração das NETs na saliva, no plasma e in vitro em indivíduos com AR e controles saudáveis e a associação com a periodontite e atividade da AR; 2) Avaliar o impacto do tratamento periodontal não cirúrgico na concentração das NETs na saliva e no plasma; 3) Investigar a associação entre a presença de polimorfismos de nucleotídeo único no gene codificador da enzima peptidil arginina deaminase 4 (PAD4) com a AR, a produção de NETs in vitro e a periodontite; 4) Sistematizar as evidências disponíveis na literatura sobre o efeito do tratamento periodontal não cirúrgico sobre os principais parâmetros clínicos e laboratoriais da AR e score de atividade da doença 28 (DAS28). Material e Métodos: Para atender aos objetivos 1 e 2, a concentração de NETs na saliva, no plasma e na cultura de neutrófilos isolados do sangue periférico foi determinada por meio da identificação do complexo mieloperoxidase (MPO)-DNA com o uso do kit PicoGreen®. Para atender ao objetivo 3 foi realizada a extração do DNA genômico das células mononucleares do sangue periférico de indivíduos com AR e controles e foi realizada a genotipagem para os polimorfismos de nucleotídeo único PADI4_89, PADI4_90, PADI4_92 e PADI_104. Para atender ao objetivo 4 foi realizada uma overview incluindo revisões sistemáticas que avaliaram o efeito do tratamento periodontal não cirúrgico sobre os parâmetros da AR. A busca foi realizada nas principais bases de dados, sem restrição de idioma ou data de publicação. Foi realizada ainda uma meta-análise incluindo dados dos estudos primários identificados nas revisões sistemáticas analisadas. Resultados: 1) e 2) Indivíduos com AR e com periodontite apresentaram maior concentração de NETs na saliva, no plasma e in vitro. O tratamento periodontal não cirúrgico reduziu a concentração de NETs na saliva e plasma de indivíduos com AR. 3) Não foi observada associação entre a presença de genótipos polimórficos e a AR. A presença de um haplótipo homozigoto para o polimorfismo foi associada a uma maior produção de NETs in vitro e piores parâmetros periodontais. 4) Foram incluídas na overview nove revisões sistemáticas. Os principais desfechos avaliados foram DAS28; proteína C-Reativa e/ou velocidade de hemossedimentação. A meta-análise mostrou que o tratamento periodontal não cirúrgico resultou em diminuição significativa do DAS28. Conclusão: A concentração das NETs na saliva, no plasma e na cultura de neutrófilos de sangue periférico está associada a AR e a periodontite, podendo representar o elo entre as duas doenças. O tratamento periodontal não cirúrgico leva à redução da atividade da AR. Polimorfismos no gene PADI4 estão associados a produção de NETs in vitro e à presença de periodontite.

Introduction: Neutrophil extracellular traps (NETs) are recognized as one of the relevant mechanisms in the pathogenesis of periodontitis and rheumatoid arthritis (RA). However, the participation of NETs as a linking mechanism between the two diseases remains poorly understood. Objectives: 1) To investigate the concentration of NETs in saliva, plasma, and in vitro in individuals with RA and healthy controls and the association of NETs with periodontitis and RA activity; 2) To evaluate the impact of non-surgical periodontal treatment on the concentration of NETs in saliva and plasma; 3) To investigate the association between the presence of single nucleotide polymorphisms in the gene coding for the enzyme peptidyl arginine deaminase 4 (PAD4) with RA, in vitro production of NETs, and periodontitis; 4) To systematize the evidence available in the literature on the effect of non-surgical periodontal treatment on the main clinical and laboratory parameters of RA and disease activity score 28 (DAS28). Material and Methods: To accomplish Objectives 1 and 2, the concentration of NETs in saliva, plasma, and culture of neutrophils isolated from peripheral blood was determined by identifying the myeloperoxidase (MPO)-DNA complex using the PicoGreen kit®. To accomplish Objective 3, genomic DNA was extracted from peripheral blood mononuclear cells of individuals with RA and healthy controls, and genotyping was performed for single nucleotide polymorphisms PADI4_89, PADI4_90, PADI4_92, and PADI_104. To accomplish the Objective 4, an overview, including systematic reviews that evaluated the effect of non-surgical periodontal treatment on RA parameters, was performed. The search was carried out in the main databases, with no restriction on language or date of publication. A meta- analysis, including data from the primary studies identified in the analyzed systematic reviews, was also performed. Results: For Objectives 1 and 2, individuals with RA and periodontitis showed a higher concentration of NETs in saliva, plasma, and in vitro. Non-surgical periodontal treatment reduced the concentration of NETs in saliva and plasma of individuals with RA. For Objective 3, no association between the presence of polymorphic genotypes and RA was observed. The presence of a homozygous haplotype for the polymorphism was associated with a higher production of NETs in vitro and worse periodontal parameters. For Objective 4, nine systematic reviews were included in the overview. The main outcomes evaluated were DAS28, C-Reactive protein, and/or erythrocyte sedimentation rate. The meta- analysis showed that non-surgical periodontal treatment resulted in a significant decrease in DAS28. Conclusion: The concentration of NETs in saliva, plasma and culture of peripheral blood neutrophils is associated with RA and periodontitis and may represent the link between the two diseases. Non-surgical periodontal treatment leads to reduced RA activity. Polymorphisms in the PADI4 gene are associated with the in vitro production of NETs and with presence of periodontitis.

PFKL, a novel regulatory node for NOX2-dependent oxidative burst and NETosis / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Neutrophils are predominant leukocytes in the circulation, which are essential for killing invading pathogens via the activation of effector responses and the production of reactive oxygen species (ROS), also named as "oxidative burst." When infected, activated neutrophils fight bacteria, fungi, and viruses through oxidative burst, phagocytosis, degranulation, and the production of neutrophil extracellular traps (NETs) in a neutrophil death process named as "NETosis" (Mutua and Gershwin, 2021). NETs, consisting of DNA fibers decorated with modified histones and numerous antimicrobial proteins from cytoplasmic granules and the nucleus, can either be beneficial or detrimental (Mutua and Gershwin, 2021). Several pathways can lead to this death process. In response to various stimuli, NETosis traps and clears pathogens, facilitating phagocytosis by other neutrophils and phagocytes. However, excessive NETosis often results in disease due to increasing the pro-inflammatory response and perpetuating the inflammatory condition (Hellebrekers et al., 2018; Hidalgo et al., 2019; Klopf et al., 2021). Accordingly, inhibiting aberrant NETosis may alleviate the severity of various autoimmune and inflammatory diseases.

Effect of neutrophil extracellular traps on ischemic stroke and intervention of traditional Chinese medicine / 中国中药杂志

Ischemic stroke is the leading cause of death and disability in adults in China. Recent studies have shown that neutrophil extracellular traps play a crucial role in occurrence and development of ischemic stroke. This paper reviewed the literatures on NETs since the discovery of NETs more than a decade ago, and summarized the composition of NETs, the effects of NETs on stroke, the intervention targets of NETs, and the effects of traditional Chinese medicine on NETs. NETs are an important cause of brain injury after stroke. Platelets, peptidylarginine deiminase 4, reactive oxygen species and histones are the targets to regulate NET formation in stroke. There are few researches on traditional Chinese medicine targeting NETs for stroke. Studies on the intervention of traditional Chinese medicine mainly target on neutrophils, which are the main components of NETs, and platelets, which induce the formation of NETs. The paper provided a comprehensive overview of current studies of NETs in ischemic stroke, so as to provide new ideas for the treatment and drug development of ischemic stroke.

LPS stimulating neutrophils firmly adhered to ICAM-1 to form extracellular traps depends on integrin Mac-1 and cytoskeletal proteins / 生物医学工程学杂志

Neutrophil extracellular traps (NETs) play an important role in the formation of immunothrombosis. However, how vascular endothelial cells mediate the formation of NETs has not been fully understood. We stimulated neutrophils firmly attached on the endothelial cell surface intercellular adhesion molecule-1 (ICAM-1) with lipopolysaccharide (LPS) or phorbol-12-myristate-13-acetate (PMA) for 4 h, then labeled NETs-DNA with Sytox green dye and the formation of NETs was observed by fluorescent microscopy. The area and fluorescence intensity of NETs-DNA were analyzed to quantify the formation of NETs. The results showed that both PMA and LPS were able to induce firmly adhered neutrophils on ICAM-1 to produce NETs. NETs induced by PMA were independent of neither β2 integrin lymphocyte function-associated antigen-1 (LFA-1) nor macrophage antigen complex-1 (Mac-1). In contrast, LPS-stimulated NETs were mediated by Mac-1 integrin, but not by LFA-1. After inhibition of actin filaments or Talin-1, the formation of NETs irrespective of the stimulus was significantly reduced. This study reveals the mechanism of the direct interaction between neutrophils and endothelial cells to produce NETs under inflammatory conditions, providing a new theoretical basis for the treatment of related diseases and the development of new drugs.

Formation of neutrophil extracellular traps and their effects on autoimmune diseases / 中国中药杂志

Neutrophil extracellular traps(NETs) are networks of extracellular fibers primarily composed of DNA, histones, granular proteins, and cytoplasmic proteins and released to the outside of cells by neutrophils under the stimulation of bacteria, fungi, viruses, parasites, etc. NETs are generated in two forms, suicidal NETs and vital NETs, according to different stimuli. NETs have both anti-inflammatory and pro-inflammatory effects. On the one hand, they can play the anti-microbial role to resist inflammation by capturing, fixing, and killing invading pathogens, which is a special way for neutrophils to exert host defenses. On the other hand, in case of excessive formation or insufficient elimination, they can cause tissue damage directly, and also promote the release of inflammatory factors by recruiting other pro-inflammatory cells or proteins to further expand the inflammatory response, which is related to the pathologies of many diseases. In autoimmune diseases, NETs as important sources of autoantigens, can act as danger-associated molecular patterns( DAMPs) and activate the nucleotide-binding oligomerization domain leucine-rich repeats containing pyrin domain 3(NLRP3) inflammasome and complement system, thereby breaking self-tolerance and accelerating autoimmune inflammation. In addition, NETs can also activate other immune cells(such as B cells, antigen-presenting cells, and T cells) and regulate the acquired immune response. The present study reviewed the correlation of NETs with diseases such as systemic lupus erythematosus(SLE), rheumatoid arthritis(RA), and gouty arthritis(GA) to reveal the effect of dynamic balance between formation and clearance of NETs in autoimmune diseases and provide a theoretical basis for the investigation of underlying mechanisms and targeted therapies of traditional Chinese medicine.

Neutrophil extracellular traps in autoimmune diseases / Trampas extracelulares de neutrófilos en enfermedades autoinmunes

ABSTRACT Neutrophils play an important role in immune defence against several pathogens. These cells actively participate in the innate immune response through different functions, such as chemotaxis, phagocytosis, oxidative burst and degranulation, which have been widely studied. However, in the last few years, a new function has been described; activated neutrophils are able to release web-like chromatin structures known as neutrophil extracellular traps (NETs). These structures formed by DNA, histones, and proteins, immobilize and kill microorganisms. Disruption in NET formation is associated with the pathophysiology of several disorders, including the autoimmune diseases. NETs are an important source of the autoantigens involved in the production of autoantibodies and maintenance of the inflammatory milieu. This review provides a summary of the contribution of NETs to the pathogenesis of anti-neutrophil cytoplasmic antibodies-associated vasculitis, systemic lupus erythematosus, and rheumatoid arthritis. The preliminary findings on NETs components in Sjögren.'s syndrome will also be described.

RESUMEN Los neutrófilos juegan un papel muy importante en la defensa inmune contra diferentes patógenos. Estas células participan activamente en la respuesta inmune innata a través de diferentes funciones como quimiotaxis, fagocitosis, estallido oxidativo y degranulación, las cuales han sido estudiadas ampliamente. Sin embargo, en los últimos años se ha descrito una nueva función; los neutrófilos activados son capaces de liberar redes de cromatina llamadas trampas extracelulares de neutrófilos (NETs). Estas estructuras están formadas por ADN, histonas y proteínas capaces de inmovilizar y matar microorganismos. Alteraciones en la formación de estas NETs están asociadas con la fisiopatología de varios trastornos, incluyendo las enfermedades autoinmunes (EAI). Las NETs son consideradas una fuente de autoantígenos que ayudan a la producción de autoanticuerpos y al mantenimiento de un ambiente inflamatorio. Esta revisión resume la contribución de las NETs a la patogénesis de vasculitis asociada a anticuerpos contra el citoplasma de los neutrófilos, lupus eritematoso sistémico y artritis reumatoide. Adicionalmente, se describirán los resultados preliminares de la detección de componentes de las NETs en pacientes con síndrome de Sjögren.

Autophagy in neutrophils

Autophagy is a highly conserved intracellular degradation and energy-recycling mechanism that contributes to the maintenance of cellular homeostasis. Extensive researches over the past decades have defined the role of autophagy innate immune cells. In this review, we describe the current state of knowledge regarding the role of autophagy in neutrophil biology and a picture of molecular mechanism underlying autophagy in neutrophils. Neutrophils are professional phagocytes that comprise the first line of defense against pathogen. Autophagy machineries are highly conserved in neutrophils. Autophagy is not only involved in generalized function of neutrophils such as differentiation in bone marrow but also plays crucial role effector functions of neutrophils such as granule formation, degranulation, neutrophil extracellular traps release, cytokine production, bactericidal activity and controlling inflammation. This review outlines the current understanding of autophagy in neutrophils and provides insight towards identification of novel therapeutics targeting autophagy in neutrophils.

Research progress on neutrophil extracellular traps in tumor / 浙江大学学报·医学版

Neutrophil extracellular traps(NET)is neutrophil-derived extracellular fiber web-like structure, composed of DNA scaffold studded with various active proteins. In addition to its bactericidal effect, NET is closely related to various diseases including immune disease, thrombosis and tumor. Recently, lots of researches have shown that NET is highly expressed in a variety of tumors, tumor cells and microenvironment can promote NET formation, whereas NET participates in tumor progression as well, and is closely related to tumor proliferation, metastasis and thrombosis, which provides new clinical thinking in tumor diagnosis as well as treatment indeed. This review will focus on the research progress of NET and tumor, meanwhile make a prospect for its clinical application value.

Characteristics of neutrophil extracellular traps in patients with periodontitis and gingivitis

Abstract We sought to compare the characteristics and clinical significance of neutrophil extracellular traps in gingival samples from patients with periodontitis and those with gingivitis. The clinical indexes of gingival samples from patients with periodontitis and gingivitis were measured; the expression of TNF-alpha and IL-8 was measured by real-time fluorescent quantitative PCR; and the expression of TLR-8 and MMP-9 was measured by western blotting assays. Chemotaxis, phagocytosis and phagocytic activity of neutrophils were measured. Compared with the healthy group, the expression of TNF-α and IL-8 in the periodontitis group and the gingivitis group increased significantly (p < 0.05), and TNF-α in the gingivitis group was significantly lower than that in the healthy group (p < 0.05). The expression of IL-8 in the periodontitis group was significantly higher than that in the periodontitis group (p < 0.05). Furthermore, the expression of TLR-8 and MMP-9 in the periodontitis group was different from that in the gingivitis group and the healthy group, and the expression of TLR-8 and MMP-9 in the gingivitis group was significantly different from that in the healthy group (p < 0.05). In addition, the neutrophil mobility index in healthy people was 3.02 ± 0.53, that in the periodontitis group was 2.21 ± 0.13, and that in the gingivitis group was 2.31 ± 0.12. In conclusion, the chemotaxis of neutrophils in gingival samples of patients with periodontitis and gingivitis was decreased, the phagocytotic ability and activity of neutrophils were reduced, and the release of the extracellular trap-releasing inducible factors TNF-alpha and IL-8 also declined.

Could neutrophil extracellular traps be the new prognostic markers of cancer?

ABSTRACT Neutrophil extracellular traps (NETs) were described more than one decade ago, but recently, the interest in these structures has increased due to their involvement in cancer progression, cancer-related thrombosis, and development of metastasis. This protumoral role of NETs strengthens their potential as new prognostic markers of cancer.

Armadilhas extracelulares dos neutrófilos: descrição e envolvimento em processos autoimunes / Neutrophil extracellular traps: description and autoimmune disease involvement

Somando a maioria dos leucócitos presentes na corrente sanguínea, os neutrófilos são células polimorfonucleares e especializadas no combate a infecções através da fagocitose do patógeno e liberação de seus conteúdos granulares. Também são capazes de liberar armadilhas extracelulares (neutrophil extracellular traps, NETs), que são estruturas constituídas de componentes intracelulares, como fibras de cromatina e proteínas derivadas de grânulos citoplasmáticos. As NETs são liberadas em resposta a vários estímulos, como de microrganismos, citocinas e complexos antígeno-anticorpo, e possuem o papel de capturar microrganismos e até mesmo matá-los. Porém, quando essas estruturas não são completamente eliminadas pelo organismo, elas podem gerar danos à saúde, pois podem ser produzidos anticorpos contra as estruturas que as compõem. Neste artigo, é feita uma revisão dos acontecimentos que levam à liberação de NETs pelos neutrófilos, a importância disso para a saúde e o envolvimento dessas estruturas em processos de autoimunidade, utilizando artigos publicados desde a descoberta das NETs, que ocorreu em 2004.

Accounting for most leukocytes present in the bloodstream, neutrophils are polymorphonuclear cells that specialize in dealing with microorganisms through phagocytosis and release of granule contents. They are also able to release extracellular traps (NETs), which are structures consisting of intracellular components such as chromatin fibers and proteins derived from cytoplasmic granules. NETs are released in response to various stimuli, such as to microorganisms, cytokines and immune complexes, and their role is to capture microorganisms and kill them. However, when these structures are not properly eliminated by the body, they may generate health damage because antibodies can be produced against them. This article provides a review of the events triggering the release of NETs, their importance for health and the involvement of these structures in autoimmune processes, using articles published since the discovery of NETs in 2004.

Significance of neutrophil extracellular trap and its markers in the early diagnosis of community-acquired pneumonia in children / 中国当代儿科杂志

OBJECTIVE@#To study the significance of plasma neutrophil extracellular trap (NET) and its markers in the diagnosis of community-acquired pneumonia (CAP) in children.@*METHODS@#A total of 160 children with CAP were enrolled as the CAP group, and 50 healthy children were enrolled the control group. According to disease severity, the CAP group was further divided into a mild CAP subgroup with 137 children and a severe CAP subgroup with 23 children. According to the pathogen, the CAP group was further divided into a bacterial pneumonia subgroup with 78 children, a Mycoplasma pneumonia subgroup with 35 children, and a viral pneumonia subgroup with 47 children. The levels of plasma NET and its markers [antibacterial peptide (LL-37), extracellular free DNA (cfDNA), and deoxyribonuclease I (DNase I)] were measured. Receiver operating characteristic (ROC) curve was used to analyze the value of each index in diagnosing CAP and assessing its severity.@*RESULTS@#Compared with the control group, the CAP group had significant increases in the levels of NET, LL-37, and cfDNA and a significant reduction in the activity of DNase I (P0.05). In the CAP group, plasma NET levels were positively correlated with white blood cell count (WBC), percentage of neutrophils, and serum levels of C-reactive protein (CRP), procalcitonin and tumor necrosis factor-α (r=0.166, 0.168, 0.275, 0.181 and 0.173 respectively, P<0.05); LL-37 and cfDNA levels were positively correlated with WBC (r=0.186 and 0.338 respectively, P<0.05) and CRP levels (r=0.309 and 0.274 respectively, P<0.05); the activity of DNase I was negatively correlated with CRP levels (r=-0.482, P<0.05). The ROC curve analysis showed that NET, LL-37, cfDNA, and DNase I had an area under the ROC curve (AUC) of 0.844, 0.648, 0.727, and 0.913 respectively in the diagnosis of CAP, with optimal cut-off values of 182.89, 46.26 ng/mL, 233.13 ng/mL, and 0.39 U/mL respectively, sensitivities of 88.12%, 35.63%, 54.37%, and 91.25% respectively, and specificities of 74.00%, 92.00%, 86.00%, and 76.00% respectively. In the assessment of the severity of CAP, NET, LL-37, cfDNA, and DNase I had an AUC of 0.873, 0.924, 0.820, and 0.778 respectively, with optimal cut-off values of 257.7, 49.11 ng/mL, 252.54 ng/mL, and 0.29 U/mL respectively, sensitivities of 83.21%, 86.96%, 78.26%, and 95.65% respectively, and specificities of 78.26%, 83.94%, 76.64%, and 56.93% respectively.@*CONCLUSIONS@#Plasma NET and its related markers have a certain value in diagnosing CAP and assessing its severity in children.

Respiratory syncytial virus increases eosinophil extracellular traps in a murine model of asthma

BACKGROUND: Respiratory viral infections are the leading cause of asthma exacerbations. Eosinophil activation results in the formation of eosinophil extracellular traps (EETs), which release web-like structures of DNA and proteins that bind, disarm and extracellularly kill pathogens. OBJECTIVE: We investigated whether the respiratory syncytial virus (RSV) in vitro could induce EETs in bronchoalveolar lavage fluid eosinophils in a murine model of asthma. METHODS: BALB/cJ mice (6–8 weeks old) were sensitized with 2 subcutaneous injections of ovalbumin (20 μg) on days 0 and 7, followed by three intranasal challenges with ovalbumin (100 μg) on days 14, 15, and 16 of the protocol. The control group received Dulbecco's phosphate-buffered saline. Bronchoalveolar lavage fluid eosinophils of ovalbumin group or control group were stimulated with RSV (103 PFU/mL) in vitro for 3 hours. After that, culture supernatant was collected to perform the analyses proposed in this study. RESULTS: We verified an increase in extracellular DNA concentration in bronchoalveolar lavage fluid eosinophils from ovalbumin group stimulated with RSV (10³ PFU/mL) in vitro, which was confirmed by confocal microscopy. We demonstrated that most cells are negative for annexin V and propidium iodide in all groups evaluated. Also, RSV in vitro decreased interferon-ɣ in culture supernatant when compared to the ovalbumin group. CONCLUSION: In this study, we demonstrated for the first time that RSV in vitro induces EETs formation in eosinophils from asthmatic mice.

Immune mechanisms in the different phases of acute tubular necrosis

Acute kidney injury is a clinical syndrome that can be caused by numerous diseases including acute tubular necrosis (ATN). ATN evolves in several phases, all of which are accompanied by different immune mechanisms as an integral component of the disease process. In the early injury phase, regulated necrosis, damage-associated molecular patterns, danger sensing, and neutrophil-driven sterile inflammation enhance each other and contribute to the crescendo of necroinflammation and tissue injury. In the late injury phase, renal dysfunction becomes clinically apparent, and M1 macrophage-driven sterile inflammation contributes to ongoing necroinflammation and renal dysfunction. In the recovery phase, M2-macrophages and anti-inflammatory mediators counteract the inflammatory process, and compensatory remnant nephron and cell hypertrophy promote an early functional recovery of renal function, while some tubules are still badly injured and necrotic material is removed by phagocytes. The resolution of inflammation is required to promote the intrinsic regenerative capacity of tubules to replace at least some of the necrotic cells. Several immune mechanisms support this wound-healing-like re-epithelialization process. Similar to wound healing, this response is associated with mesenchymal healing, with a profound immune cell contribution in terms of collagen production and secretion of profibrotic mediators. These and numerous other factors determine whether, in the chronic phase, persistent loss of nephrons and hyperfunction of remnant nephrons will result in stable renal function or progress to decline of renal function such as progressive chronic kidney disease.