Research advances in neutrophil extracellular traps and liver diseases / 临床肝胆病杂志

Neutrophils play an immune defense role by releasing the proteases such as neutrophil elastase and myeloperoxidase to form neutrophil extracellular trap （NET） and participate in the inflammatory response of various liver diseases， but the excessive release of NET may worsen liver tissue damage and has thus become one of the risk factors for liver diseases. In recent years， studies have shown that the excessive release of NET can promote the progression of liver diseases （such as viral hepatitis， nonalcoholic steatohepatitis， and hepatic ischemia-reperfusion injury） to liver cancer， and clarifying the mechanism of action of NET is of great importance for the diagnosis and progression of liver diseases. Therefore， this article elaborates on the latest research advances in NET in liver diseases， so as to provide new insights into the diagnosis and treatment of liver diseases and the prevention of liver cancer.

Rol de trampas extracelulares de neutrófilos en el pronóstico de las infecciones del sistema respiratorio / Role of neutrophil extracellular traps in the prognosis of respiratory tract infections

Las trampas extracelulares de neutrófilos (NET, por sus siglas en inglés) han surgido recientemente como un vínculo potencial entre la inmunidad y la inflamación, que podría cumplir un papel clave en la patogénesis de las infecciones de vías respiratorias. El objetivo de esta revisión es determinar su rol como marcador pronóstico en enfermedades infecciosas de vías respiratorias. Para la elaboración de este artículo de revisión narrativa se consultaron las publicaciones disponibles a través de una búsqueda automatizada en las bases de datos de PubMed, Scopus y Embase. Las concentraciones elevadas de trampas extracelulares de neutrófilos (cfADN, complejos de mieloperoxidasas-ADN) en pacientes con cuadro clínico grave por infecciones de vías respiratorias, se relacionan con una estancia hospitalaria más larga, periodo prolongado de administración de antibióticos, aumento del riesgo de ingreso a la UCI, necesidad de ventilación mecánica, disfunción orgánica e incluso la muerte (p ≤ 0,05). A pesar de no contar con un parámetro de medición estandarizado, el exceso de trampas extracelulares de neutrófilos se corresponde con la gravedad del daño tisular observado en pacientes con infecciones de vías respiratorias, esto revela el importante rol pronóstico de la respuesta de los neutrófilos y del proceso de la NETosis en las enfermedades infecciosas pulmonares

Neutrophil extracellular traps (NET) have recently emerged as a potential link between immunity and inflammation, which could play a key role in the pathogenesis of respiratory tract infections. This review aims to determine the role of neutrophil extracellular traps as prognostic markers in respiratoria tract infectious diseases. For this article a literature review was undertaken, consulting available publications through an automated search in PubMed, Scopus, and Embase databases. High concentrations of neutrophil extracellular traps (cfDNA, Myeloperoxidase-DNA complexes) in patients with severe clinical presentation due to respiratory tract infections are related to a longer length of hospital stay, prolonged period of antibiotic administration and increased risk of admission to the ICU, need for mechanical ventilation, organ dysfunction and even death (p ≤ 0.05). Despite not having a standardized measurement parameter, the excess of neutrophil extracellular traps corresponds to the severity of tissue damage observed in patients with respiratory tract infections, revealing the important prognostic role of the neutrophil response and NETosis process in pulmonary infectious diseases

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.

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.

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.

Pancreatic melatonin enhances anti-tumor immunity in pancreatic adenocarcinoma through regulating tumor-associated neutrophils infiltration and NETosis

Tumor microenvironment contributes to poor prognosis of pancreatic adenocarcinoma (PAAD) patients. Proper regulation could improve survival. Melatonin is an endogenous hormone that delivers multiple bioactivities. Here we showed that pancreatic melatonin level is associated with patients' survival. In PAAD mice models, melatonin supplementation suppressed tumor growth, while blockade of melatonin pathway exacerbated tumor progression. This anti-tumor effect was independent of cytotoxicity but associated with tumor-associated neutrophils (TANs), and TANs depletion reversed effects of melatonin. Melatonin induced TANs infiltration and activation, therefore induced cell apoptosis of PAAD cells. Cytokine arrays revealed that melatonin had minimal impact on neutrophils but induced secretion of Cxcl2 from tumor cells. Knockdown of Cxcl2 in tumor cells abolished neutrophil migration and activation. Melatonin-induced neutrophils presented an N1-like anti-tumor phenotype, with increased neutrophil extracellular traps (NETs) causing tumor cell apoptosis through cell-to-cell contact. Proteomics analysis revealed that this reactive oxygen species (ROS)-mediated inhibition was fueled by fatty acid oxidation (FAO) in neutrophils, while FAO inhibitor abolished the anti-tumor effect. Analysis of PAAD patient specimens revealed that CXCL2 expression was associated with neutrophil infiltration. CXCL2, or TANs, combined with NET marker, can better predict patients' prognosis. Collectively, we discovered an anti-tumor mechanism of melatonin through recruiting N1-neutrophils and beneficial NET formation.

Memantine hydrochloride promotes the antibacterial effect of neutrophils against methicillin-resistant Staphylococcus aureus and the related mechanism / 中华微生物学和免疫学杂志

Objective:To investigate whether memantine hydrochloride (MEM) could promote the bactericidal effect of neutrophils against methicillin-resistant Staphylococcus aureus (MRSA) and the possible mechanism. Methods:Neutrophils were co-incubated with different concentrations of MEM and MRSA for 4 h. Then the cell lysates were collected and cultured on plate for survival bacteria counting. After co-incubation, the neutrophils were collected to detect the production of reactive oxygen species (ROS) and the release of neutrophil extracellular traps (NETs). A mouse model of MRSA infection was established, and then the mice were treated with or without MEM. Blood, spleen and kidney samples were collected from the mice for bacterial colony counting and blood procalcitonin (PCT) detection. In the 48 h survival experiment, the mice were first infected with MRSA, and then treated with MEM or PBS. The survival rates of the mice were calculated and the survival curves were drawn.Results:The number of MRSA co-cultured with neutrophils decreased significantly in the presence of MEM, and within a certain concentration range, the survival number of MRSA decreased with the increase of MEM concentration. Moreover, MEM could significantly promote the production of ROS by neutrophils and the formation of NETs. In vivo experiment showed that the concentration of PCT in mouse blood samples was lower in the MRSA+ MEM group than in the MRSA+ PBS group. The animal experiment also revealed that MEM significantly decreased the bacteria loads in mouse blood and organs and increased the 48 h survival rate after MRSA infection.Conclusions:MEM could significantly promote the bactericidal effect of neutrophils against MRSA, which might be related to the enhanced generation of ROS by neutrophils and the formation of NETs.

Correlation of neutrophil extracellular traps with T cells differentiation in Hashimoto′s thyroiditis mice and vitamin D′s relief effect / 中华内分泌代谢杂志

Objective:To investigate correlation between neutrophil extracellular traps(NETs) formation and T cell subsets in mice with experimental autoimmune thyroiditis(EAT) and the impact of active vitamin D intervention.Methods:Six-week-old female BALB/c mice were randomly divided into Control group, EAT group and 1, 25 dihydroxy vitamin D 3[1, 25(OH) 2D 3] treatment group(VitD group; n=6/group). HE staining was used to observe thyroid pathology. Plasma thyroglobulin antibody(TGAb), thyroid peroxidase antibody(TPOAb), and 1, 25(OH) 2D 3 were measured by ELISA. Peripheral NETs formation, Th1, Th2, and Th17 cell ratio from spleen were measured by flow cytometry. Correlation between NETs formation rate and Th1, Th2, and Th17 cell ratio was analyzed. Results:Compared with Control group, mice in EAT group had significantly increased thyroid inflammation scores, thyroiditis morbidity, TPOAb, TGAb levels, NETs formation rate, Th2(CD4 + IL-4 + or CD4 + IL-13 + )and Th17 cell proportions( P were <0.001, 0.002, 0.007, <0.001, <0.001, 0.003, 0.001, and 0.002, respectively), and significant decreased 1, 25(OH) 2D 3, Th1 cell proportions, Th1/Th2(CD4 + IL-4 + ), Th1/Th2(CD4 + IL-13 + ), and Th1/Th17 ratios( P were 0.010, 0.018, 0.010, 0.005, and 0.007, respectively). Compared with the EAT group, the VitD group had lower thyroid inflammation scores, TPOAb, TGAb levels, NETs formation rate, Th2(CD4 + IL-4 + or CD4 + IL-13 + ) and Th17 cell proportions( P were 0.044, 0.007, <0.001, 0.001, 0.014, 0.008, and 0.001, respectively), and significant higher Th1 cell ratio, Th1/Th2(CD4 + IL-13 + ) and Th1/Th17 ratio( P were 0.011, 0.009, and 0.003, respectively). The Th1/Th2(CD4 + IL-4 + ) was not significantly increased in VitD group compared with EAT group( P=0.174). NETs formation rate was positively correlated with Th2(CD4 + IL-4 + or CD4 + IL-13 + ) and Th17 cell proportion( r were 0.65, 0.59, and 0.61; and P were 0.004, 0.010, and 0.007, respectively), but not with Th1 cell proportion( r=-0.47, P=0.051). Conclusion:EAT mice were more prone to NETs formation. Active vitamin D may relieve immune imbalance with increased Th2 and Th17 cell ratio and decreased Th1 cell ratio by reducing the formation of NETs in EAT mice. Vitamin D played the protective role in thyroid by reducing thyroid pathological damage and thyroid autoantibody levels, and relived overall lymphocyte imbalance.

Effects of GSK484 on ventilator-induced lung injury and neutrophil extracelluar traps in mice / 中华麻醉学杂志

Objective:To evaluate the effects of GSK484 on ventilator-induced lung injury (VILI) and neutrophil extracelluar traps (NETs) in mice.Methods:Forty-eight SPF healthy male C57BL/6 mice, aged 5-6 weeks, weighing 15-20 g, were divided into 4 groups ( n=12 each) by a random number table method: spontaneous breathing group (group S), spontaneous breathing+ GSK484 intervention group (group SG), VILI group (group V), and VILI + GSK484 intervention group (group VG). The animals kept spontaneous breathing for 4 h after tracheal intubation in S and SG groups. The animals were mechanically ventilated for 4 h (tidal volume 30 ml/kg, respiratory rate 75 breaths/min, inspiratory/expiratory ratio 1∶2, positive end-expiratory pressure 0 mmHg, fraction of inspired oxygen 21%) in V and VG groups. At 3 days before developing the VILI model, GSK484 4 mg/kg was intraperitoneally injected once a day in SG and VG groups, while the equal volume of normal saline was given instead in S and V groups. Blood samples were collected from the abdominal aorta for blood gas analysis at 4 h of spontaneous breathing or mechanical ventilation, and PaO 2 was recorded. The mice were then sacrificed and bronchoalveolar lavage fluid (BALF) was collected and lung tissues were obtained for microscopic examination of the pathological changes (with a light microscope after HE staining) which were scored and for determination of wet to dry weight ratio (W/D ratio), concentrations of interleukin-1beta (IL-1β), IL-6, tumor necrosis factor-alpha (TNF-α) and myeloperoxidase (MPO) in BALF (by enzyme-linked immunosorbent assay), expression of peptidylarginine deiminase 4 (PAD4), neutrophil elastase (NE), high mobility group box 1 (HMGB1) and citrullinated-histone 3 (Cit-H3) in lung tissues (by Western blot). Results:Compared with S and SG groups, the lung injury score and W/D ratio were significantly increased, PaO 2 was decreased, concentrations of IL-1β, IL-6, TNF-α and MPO in BALF were increased, and the expression of PAD4, NE, HMGB1 and Cit-H3 in lung tissues was up-regulated in V and VG groups ( P<0.05). Compared with group V, the lung injury score and W/D ratio were significantly decreased, PaO 2 was increased, the concentrations of IL-1β, IL-6, TNF-α and MPO in BALF were decreased, and the expression of PAD4, NE, HMGB1 and Cit-H3 was down-regulated in group VG ( P<0.05). Conclusions:GSK484 can alleviate VILI in mice, and the mechanism is associated with inhibition of PAD4, reduction of the production of NETs and attenuation of inflammatory responses in lung tissues.

Advances in neutrophil extracellular traps in bronchopulmonary dysplasia / 国际儿科学杂志

Due to the development of neonatal intensive care, the survival rate of very preterm infants and very low birth weight infants has been significantly improved, and the incidence of bronchopulmonary dysplasia (BPD) has been obviously increasing year by year.The pathogenesis of BPD has not been clear, it is considered that inflammation is an important link in the occurrence and development of BPD at present.Neutrophils can use their neutrophil extracellular traps (NETs) to capture and kill pathogens and reduce inflammation, but excessive formation of NETs is easy to induce inflammatory imbalance, so as to damage normal cells or tissues and participate in the pathophysiological process of BPD.This paper reviews the structure, formation, function and regulatory role of NETs in BPD, and the targeted treatment strategies and potential research directions of NETs in BPD.

Research progress on neutrophil extracellular traps in oral infectious diseases / 口腔疾病防治

@#Eutrophils are the first innate immune cells to reach the site of inflammation. Neutrophils produce neutrophil extracellular traps (NETs) that can quickly capture and limit the spread of pathogens, facilitating the removal of pathogens and their debris. Neutrophils in the oral cavity are specifically transformed from circulating neutrophils in the blood, and the number of NETs released by oral neutrophils is much higher than that of circulating neutrophils, thus better maintaining the balance of the oral microenvironment. As a bimorphic fungus, only the mycelium phase of Candida albicans can induce NETs, which is related to the neutrophils' ability to sense the size of pathogenic microorganisms through neutrophil elastase. However, spherical Staphylococcus aureus are much smaller than Candida albicans, and they can still induce NETs. Porphyromonas gingivalis, as one of the microorganisms in the periodontitis complex, induces fewer NETs than Streptococcus oralis and Actinomycetes, which are two common oral microorganisms, and there may be a mechanism allowing them to escape neutrophilic immunity in the early stage of periodontitis. Although the two main pathways of NET production have been studied in detail, the mechanisms involved in the induction of NETs by different microorganisms, especially from oral neutrophils, are not well understood. This review describes the mechanism of the immune effects of pathogenic microorganisms on neutrophil NETs in the oral cavity, providing a reference for the search for therapeutic targets and the development of key drugs for treating oral infectious diseases.

The effect of extracellular vesicles derived from lung tissue on intrapulmonary inflammation and formation of neutrophil extracellular traps in sepsis rats / 中华急诊医学杂志

Objective:To investigate the effect of extracellular vesicles derived from lung tissue on intrapulmonary inflammation and the formation of neutrophil extracellular traps (NETs) in sepsis rats.Methods:Sepsis rat model was established by cecal ligation and puncture (CLP). Collagenase D and DNase I were used to dissociate the lung tissue, the impurities were removed by centrifugation, and finally, the extracellular vesicles (Ti-EVs) derived from lung tissue were separated and extracted by differential ultracentrifugation. Eighteen male SD rats were randomly divided into the sham group, sepsis group and Ti-EVs group: in the Ti-sEV group, a sepsis model was established by CLP, and Ti-EVs suspension was instilled through the airway; rats in the CLP group received CLP, and an equal volume of PBS was instilled through the airway; and rats in the sham group was treated with sham operation. The pathological changes of lung tissue were detected by hematoxylin-eosin (HE) staining after 24 h. The content of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in the bronchoalveolar lavage fluid (BALF) was measured by enzyme-linked immunosorbent assay (ELISA). Immunofluorescence was used to detect the NETs content in lung tissue.Results:The isolated extracellular vesicles derived from rat lung tissue were observed by transmission electron microscopy as double-layer circular cystic vesicles with particle diameter mainly distributed at 150 nm. Western blot showed positive expression of EVs markers CD9, CD63, and Tsg101. HE staining of lung tissue showed alveolar integrity damage and a large number of inflammatory cells infiltrated in the lung of sepsis rats. Compared with the CLP group, the degree of lung tissue damage was more serious in the Ti-EVs group and the levels of IL-1β, TNF-α and IL-6 in the BALF of rats were significantly increased ( P<0.01). The formation of NETs in the lungs of the rats in the sepsis group and the Ti-EVs group was observed under the laser confocal microscope. Compared with the sepsis group, the fluorescence intensity of NETs in the lung tissues of the Ti-EVs group increased significantly. Conclusions:After enzymatic digestion, differential ultracentrifugation and other treatments, the extracellular vesicles derived from rat lung tissue with high purity can be successfully isolated and extracted. In the process of septic lung injury, extracellular vesicles in lung tissue can aggravate the inflammatory response in the lung and promote the formation of NETs.

Self-assembling, pH-responsive nanoflowers for inhibiting PAD4 and neutrophil extracellular trap formation and improving the tumor immune microenvironment

Self-assembling carrier-free nanodrugs are attractive agents because they accumulate at tumor by an enhanced permeability and retention (EPR) effect without introduction of inactive substances, and some nanodrugs can alter the immune environment. We synthesized a peptidyl arginine deiminase 4 (PAD4) molecular inhibitor, ZD-E-1M. It could self-assembled into nanodrug ZD-E-1. Using confocal laser scanning microscopy, we observed its cellular colocalization, PAD4 activity and neutrophil extracellular traps (NETs) formation. The populations of immune cells and expression of immune-related proteins were determined by single-cell mass cytometry. ZD-E-1 formed nanoflowers in an acidic environment, whereas it formed nanospheres at pH 7.4. Accumulation of ZD-E-1 at tumor was pH-responsive because of its pH-dependent differences in the size and shape. It could enter the nucleus and bind to PAD4 to prolong the intracellular retention time. In mice, ZD-E-1 inhibited tumor growth and metastasis by inhibiting PAD4 activity and NETs formation. Besides, ZD-E-1 could regulate the ratio of immune cells in LLC tumor-bearing mice. Immunosuppressive proteins like LAG3 were suppressed, while IFN-γ and TNF-α as stimulators of tumor immune response were upregulated. Overall, ZD-E-1 is a self-assembling carrier-free nanodrug that responds to pH, inhibits PAD4 activity, blocks neutrophil extracellular traps formation, and improves the tumor immune microenvironment.

Role of eosinophil extracellular traps in the pathogenesis of chronic airway diseases / 中华微生物学和免疫学杂志

Eosinophil extracellular traps (EETs), an important pathway of eosinophil to exert its effects, are composed of DNA fibers, histone and eosinophil granule proteins. Recently, many researches have shown that EETs play an important role in the genesis and development of respiratory diseases including asthma, allergic bronchopulmonary aspergillosis and chronic obstructive pulmonary disease. EETs can directly damage airway epithelial cells, promote airway inflammation and airway hypersecretion, increase the stickiness of secretions and induce the generation of autoantibody, helping eosinophils and their products participate in a cascade of events leading to inflammation and disease. Researches on EETs can also be helpful in investigating new targets for the treatment of chronic airway diseases.

A preliminary study of the role of neutrophil extracellular traps in patients with primary biliary cholangitis / 临床肝胆病杂志

Objective To investigate the expression level of neutrophil extracellular traps (NET) in the peripheral blood and liver tissue of primary biliary cholangitis (PBC) patients and its correlation with clinical biochemical parameters. Methods A total of 24 PBC patients who were admitted to Renji Hospital, Shanghai Jiao Tong University School of Medicine, from August 2016 to August 2020 were enrolled, as well as 8 patients with primary sclerosing cholangitis (PSC) and 19 patients with autoimmune hepatitis (AIH) matched for age, and 19 healthy individuals were enrolled as healthy control group (HC group). The serum level of myeloperoxidase (MPO) was measured, and its correlation with clinical indices were analyzed. Immunofluorescence assay was used to measure the expression of NET in the liver of PBC patients, and an in vitro experiment was to compare the ability of peripheral blood neutrophils to produce NET between PBC patients and healthy controls. Normally distributed continuous data were expressed as mean±standard deviation, and the independent samples t -test was used for comparison between two groups; for the non-normally distributed continuous data expressed as M ( P 25 - P 75 ), the Kruskal-Wallis H test was used for comparison between multiple groups, and the Mann-Whitney U test was used for comparison between two groups. A correlation analysis was performed for MPO level and liver-related laboratory markers, and the Spearman's correlation coefficient was calculated. Results The serum level of MPO in the PBC group was increased to 811.21 (450.67-1 216.20) ng/mL, which was significantly higher than that in the AIH group [468.58 (142.63-812.43) ng/mL] and the HC group [357.54 (203.52-811.21) ng/mL] ( P < 0.05), suggesting that there was a significant increase in the production of NET in peripheral blood of PBC patients. The PSC patients had a serum MPO level of 763.56 (489.59-1 633.14) ng/mL, which was significantly higher than that in the HC group ( P < 0.05). MPO level was positively correlated with alkaline phosphatase ( r =0.500, P < 0.05), gamma-glutamyl transpeptidase ( r =0.426, P < 0.05), alanine aminotransferase ( r =0.521, P < 0.05), and aspartate aminotransferase ( r =0.547, P < 0.01). Confocal immunofluorescence showed colocalization of H3Cit and MPO in the liver of PBC patients. In vitro experiment showed that compared with the HC group, the PBC group had an increase in NET produced by peripheral blood neutrophils after in vitro stimulation and an increase in spontaneous production of NET. Conclusion There is an increase in NET in peripheral blood and liver of PBC patients, and the content of peripheral blood NET is positively correlated with biochemical parameters of liver function. NET may become a novel biomarker for assessing the severity of PBC.

Effects of Cholesterol-lowering Agents on Proliferation, Invasion and Neutrophil Extracellular Trap Formation in Liver Cancer Cells / 肿瘤防治研究

Objective To investigate the effects of cholesterol-lowering agents on the proliferation, stemness characters, migration, invasion, and neutrophil extracellular traps formation (NETs) formation in liver cancer cells. Methods ASPP2 or HMGCR gene was knocked down in mouse liver cancer cell Hepa1-6 to establish cells with high or low cholesterol, respectively. Simvastatin and berberine were used to reduce cholesterol synthesis. CCK-8 and plate cloning assays were conducted to detect the proliferation ability of liver cancer cells. Sphere formation assay and qRT-PCR were used to analyze the stemness character and expression of related genes. Wound-healing assay and Transwell assay were used to analyze the ability of cell migration and invasion. Immunofluorescence staining was carried out to analyze the effect of lipid-lowering agent on NETs formation. Results Cholesterol-lowering agents significantly inhibited the proliferation and stemness-related gene expression of Hepa1-6 cells (P < 0.001), significantly inhibited the migration and invasion of Hepa1-6 cells (P < 0.001), and significantly inhibited the neutrophil-induced invasion and formation of NETs (P < 0.001). Conclusion Cholesterol-lowering agents suppress the proliferation and invasion via inhibiting the stemness characters and NETs formation in liver cancer cells. It is a potential strategy for the treatment of liver cancer metastasis.

Research progress in neutrophil extracellular traps in obstetrics / 中华围产医学杂志

Neutrophils plays an essential role in innate immunity. Neutrophil extracellular traps (NETs) are released by activated neutrophils in response to infection. However, excessive NETs can result in body dysfunction and organ damage and participate in several pathological conditions. This paper mainly focuses on the formation, regulatory mechanism and role of NETs in obstetric diseases, such as preeclampsia, gestational diabetes mellitus and preterm birth, and listed the current medications used to block the formation of NETs in obstetrics.

Effects of neutrophil extracellular traps on proliferation and apoptosis of human amniotic epithelial cells and its possible mechanisms / 中华围产医学杂志

Objective:To investigate the effects of neutrophil extracellular traps (NETs) on the proliferation and apoptosis of human amniotic epithelial cells.Methods:NETs were induced in vitro from the neutrophil cells obtained from the peripheral blood of normal pregnant women before elective cesarean section at full-term. Human amniotic epithelial cell lines (WISH cells) were cultured in vitro, and were divided into four groups:(1) control group: without any stimulus; (2) NETs group: WISH cells were stimulated with NETs (500 ng/ml); (3) NETs+SB203580 (p38 kinase inhibitor) group: WISH cells were pretreated with SB203580 (5 μmol/L) for 30 min and then NETs (500 ng/ml) was added; (4) SB203580 group: only SB203580 was added. After stimulating for 48 h, cell proliferation assay, lactate dehydrogenase(LDH) assay, and flow cytometry assay were used to detect the cell proliferation rate, LDH level of cell supernatant, and cell apoptosis rate among different groups. The results were analyzed and compared using one-way analysis of variance and LSD- t test. Results:(1) Cell proliferation: The cell proliferation ratio in the NETs group was lower than that in the control group [(9.379±0.775)% vs (36.560±1.208)%, LSD- t=20.78, P<0.001]; and the figure in the NETs+SB203580 group [(27.920±0.926)%] was higher than that in the NETs group (LSD- t=14.18, P<0.001). (2)LDH: There was an increased LDH level in the cell supernatant of the NETs group compared with the control group (1.518±0.038 vs 0.274±0.004, LSD -t=44.25, P<0.05), and the LDH level in the NETs+SB203580 group (0.857±0.009) was decreased than that in the NETs group (LSD -t=23.51, P<0.001). (3) Apoptosis: Compared with the control group, the cell apoptosis level of the NETs group was increased [(14.290±0.141)% vs (10.110±0.044)%, LSD- t=21.76, P<0.001]; but that in the NETs+SB203580 group [(10.500±0.218)%] was lower than in the NETs group (LSD- t=19.70, P<0.001). Conclusion:p38/mitogen-activated protein kinases signaling pathway may be involved in the process of NETs, inhibiting proliferation and promoting apoptosis of human amniotic epithelial cells.

Research progress of neutrophil extracellular traps in the pathogenesis of ischemic stroke / 中华神经科杂志

Ischemic stroke is one of the primary causes of death and disability worldwide. Neutrophils can release depolymerized chromatin and proteins to form neutrophil extracellular traps (NETs) and participate in intravascular thrombus formation. In recent years, NETs have received increasing attention in the study of acute ischemic stroke. The results indicate that NETs play an important role in the pathogenesis of acute ischemic stroke. This review presented recent advances in the study of NETs in acute ischemic stroke.

Research progress of neutrophil extracellular traps in rheumatic diseases / 中华实用儿科临床杂志

Rheumatic diseases are inflammatory diseases characterized by severe immune dysregulation, which can affect tissues and joints.Neutrophils are the key factor contributing to immune disorders, and they play an important role in rheumatic diseases.Neutrophil extracellular traps (NETs) are network structures released by neutrophil granulocytes when stimulated.As a novel immune defense mechanism, NETs have attracted wide attention.In this paper, the formation, function and role of NETs in rheumatic diseases and other diseases were reviewed.