Recombinant thrombomodulin and recombinant antithrombin attenuate pulmonary endothelial glycocalyx degradation and neutrophil extracellular trap formation in ventilator-induced lung injury in the context of endotoxemia.

BACKGROUND: Vascular endothelial damage is involved in the development and exacerbation of ventilator-induced lung injury (VILI). Pulmonary endothelial glycocalyx and neutrophil extracellular traps (NETs) are endothelial protective and damaging factors, respectively; however, their dynamics in VILI and the effects of recombinant thrombomodulin and antithrombin on these dynamics remain unclear. We hypothesized that glycocalyx degradation and NETs are induced by VILI and suppressed by recombinant thrombomodulin, recombinant antithrombin, or their combination. METHODS: VILI was induced in male C57BL/6J mice by intraperitoneal lipopolysaccharide injection (20 mg/kg) and high tidal volume ventilation (20 mL/kg). In the intervention groups, recombinant thrombomodulin, recombinant antithrombin, or their combination was administered at the start of mechanical ventilation. Glycocalyx degradation was quantified by measuring serum syndecan-1, fluorescence-labeled lectin intensity, and glycocalyx-occupied area in the pulmonary vascular lumen. Double-stranded DNA in the bronchoalveolar fluid and fluorescent areas of citrullinated histone H3 and myeloperoxidase were quantified as NET formation. RESULTS: Serum syndecan-1 increased, and lectin fluorescence intensity decreased in VILI. Electron microscopy revealed decreases in glycocalyx-occupied areas within pulmonary microvessels in VILI. Double-stranded DNA levels in the bronchoalveolar lavage fluid and the fluorescent area of citrullinated histone H3 and myeloperoxidase in lung tissues increased in VILI. Recombinant thrombomodulin, recombinant antithrombin, and their combination reduced glycocalyx injury and NET marker levels. There was little difference in glycocalyx injury and NET makers between the intervention groups. CONCLUSION: VILI induced glycocalyx degradation and NET formation. Recombinant thrombomodulin and recombinant antithrombin attenuated glycocalyx degradation and NETs in our VILI model. The effect of their combination did not differ from that of either drug alone. Recombinant thrombomodulin and antithrombin have the potential to be therapeutic agents for biotrauma in VILI.

Neutrophil PPIF exacerbates lung ischemia-reperfusion injury after lung transplantation by promoting calcium overload-induced neutrophil extracellular traps formation.

Lung ischemia-reperfusion (I/R) injury is the main risk factor for primary graft dysfunction and patient death after lung transplantation (LTx). It is widely accepted that the main pathological mechanism of lung I/R injury are calcium overload, oxygen free radical explosion and neutrophil-mediated damage, which leading to the lack of effective treatment options. The aim of this study was to further explore the mechanisms of lung I/R injury after LTx and to provide potential therapeutic strategies. Our bioinformatics analysis revealed that the neutrophil extracellular traps (NETs) formation was closely involved in lung I/R injury after LTx, which was accompanied by up-regulation of peptidylprolyl isomerase F (PPIF) and peptidyl arginine deiminase 4 (PADI4). We further established an orthotopic LTx mouse model to simulate lung I/R injury in vivo, and found that PPIF and PADI4 inhibitors effectively reduced neutrophil infiltration, NETs formation, inflammatory response, and lung I/R injury. In the neutrophil model induced by HL-60 cell line in vitro, we found that PPIF inhibitor cyclosporin A (Cys A) better alleviated calcium overload induced inflammatory response, reactive oxygen species content and NETs formation. Further study demonstrated that interfering with neutrophil PPIF protected mitochondrial function by alleviating store-operated calcium entry (SOCE) during calcium overload and played the above positive role. On this basis, we found that the reduction of calcium content in neutrophils was accompanied by the inhibition of calcineurin (CN) and nuclear factor of activated T cells (NFAT). In conclusion, our findings suggested that neutrophil PPIF could serve as a novel biomarker and potential therapeutic target of lung I/R injury after LTx, which provided new clues for its treatment by inhibiting calcium overload-induced NETs formation.

Identification of neutrophil extracellular trap-related biomarkers in non-alcoholic fatty liver disease through machine learning and single-cell analysis.

Non-alcoholic Fatty Liver Disease (NAFLD), noted for its widespread prevalence among adults, has become the leading chronic liver condition globally. Simultaneously, the annual disease burden, particularly liver cirrhosis caused by NAFLD, has increased significantly. Neutrophil Extracellular Traps (NETs) play a crucial role in the progression of this disease and are key to the pathogenesis of NAFLD. However, research into the specific roles of NETs-related genes in NAFLD is still a field requiring thorough investigation. Utilizing techniques like AddModuleScore, ssGSEA, and WGCNA, our team conducted gene screening to identify the genes linked to NETs in both single-cell and bulk transcriptomics. Using algorithms including Random Forest, Support Vector Machine, Least Absolute Shrinkage, and Selection Operator, we identified ZFP36L2 and PHLDA1 as key hub genes. The pivotal role of these genes in NAFLD diagnosis was confirmed using the training dataset GSE164760. This study identified 116 genes linked to NETs across single-cell and bulk transcriptomic analyses. These genes demonstrated enrichment in immune and metabolic pathways. Additionally, two NETs-related hub genes, PHLDA1 and ZFP36L2, were selected through machine learning for integration into a prognostic model. These hub genes play roles in inflammatory and metabolic processes. scRNA-seq results showed variations in cellular communication among cells with different expression patterns of these key genes. In conclusion, this study explored the molecular characteristics of NETs-associated genes in NAFLD. It identified two potential biomarkers and analyzed their roles in the hepatic microenvironment. These discoveries could aid in NAFLD diagnosis and management, with the ultimate goal of enhancing patient outcomes.

Calpain-1 weakens the nuclear envelope and promotes the release of neutrophil extracellular traps.

The inducers of neutrophil extracellular trap (NET) formation are heterogeneous and consequently, there is no specific pathway or signature molecule indispensable for NET formation. But certain events such as histone modification, chromatin decondensation, nuclear envelope breakdown, and NET release are ubiquitous. During NET formation, neutrophils drastically rearrange their cytoplasmic, granular and nuclear content. Yet, the exact mechanism for decoding each step during NET formation still remains elusive. Here, we investigated the mechanism of nuclear envelope breakdown during NET formation. Immunofluorescence microscopic evaluation revealed a gradual disintegration of outer nuclear membrane protein nesprin-1 and alterations in nuclear morphology during NET formation. MALDI-TOF analysis of NETs that had been generated by various inducers detected the accumulation of nesprin-1 fragments. This suggests that nesprin-1 degradation occurs before NET release. In the presence of a calpain-1, inhibitor nesprin-1 degradation was decreased in calcium driven NET formation. Microscopic evaluation confirmed that the disintegration of the lamin B receptor (LBR) and the collapse of the actin cytoskeleton occurs in early and later phases of NET release, respectively. We conclude that the calpain-1 degrades nesprin-1, orchestrates the weakening of the nuclear membrane, contributes to LBR disintegration, and promoting DNA release and finally, NETs formation.

Eosinophil extracellular traps in respiratory ailment: Pathogenic mechanisms and clinical translation.

Background: Eosinophilic extracellular traps (EETs) are reticular complexes comprising deoxyribonucleic-Acid (DNA) fibers and granule proteins. Aims: EETs play a crucial role in antimicrobial host responses and are pathogenic when overproduced or under degraded. EETs created by eosinophils appear to enable vital immune responses against extra-cellular pathogens, nevertheless, trap overproduction is evident in pathology. Materials & Methods: As considerably research is performed, new data affirmed that EETs can alter the outcome of respiratory ailment. Results: We probe into the disclosure and specificity of EETs produced in reaction to various stimuli and propose a role for those frameworks in ailment pathogenesis and the establishment of chronic, unresolved inflammation. Discussion: Whether EETs can be used as a prospective brand-new target for the diagnosis, treatment and prognosis of respiratory ailments is a scientific theme worth studying. Conclusion: We probe into the disclosure and specificity of EETs produced in reaction to various stimuli and propose a role for those frameworks in ailment pathogenesis and the establishment of chronic, unresolved inflammation.

COVID-19 mRNA vaccines induce robust levels of IgG but limited amounts of IgA within the oronasopharynx of young children.

BACKGROUND: Understanding antibody responses to SARS-CoV-2 vaccination is crucial for refining COVID-19 immunization strategies. Generation of mucosal immune responses, including mucosal IgA, could be of potential benefit to vaccine efficacy, yet limited evidence exists regarding the production of mucosal antibodies following the administration of current mRNA vaccines to young children. METHODS: We measured the levels of antibodies against SARS-CoV-2 from a cohort of children under 5 years of age (N=24) undergoing SARS-CoV-2 mRNA vaccination (serially collected, matched serum and saliva samples) or in a convenience sample of children under 5 years of age presenting to pediatric emergency department (nasal swabs, N=103). Further, we assessed salivary and nasal samples for the ability to induce SARS-CoV-2 spike-mediated neutrophil extracellular traps (NET) formation. RESULTS: Longitudinal analysis of post-vaccine responses in saliva revealed the induction of SARS-CoV-2 specific IgG but not IgA. Similarly, SARS-CoV-2 specific IgA was only observed in nasal samples obtained from previously infected children with or without vaccination, but not in vaccinated children without a history of infection. In addition, oronasopharyngeal samples obtained from children with prior infection were able to trigger enhanced spike-mediated NET formation, and IgA played a key role in driving this process. CONCLUSIONS: Despite the induction of specific IgG in the oronasal mucosa, current intramuscular vaccines have limited ability to generate mucosal IgA in young children. These results confirm the independence of mucosal IgA responses from systemic humoral responses following mRNA vaccination and suggest potential future vaccination strategies for enhancing mucosal protection in this young age group.

TRIM8 as a predictor for prognosis in childhood acute lymphoblastic leukemia based on a signature of neutrophil extracellular traps.

Background: Neutrophil extracellular traps (NETs) can be attributed to the metastasis, occurrence, and immune evasion of cancer cells. We investigated the prognostic value of NET-related genes in childhood acute lymphoblastic leukemia (cALL) patients. Methods: Differential gene expression analysis was conducted on samples collected from public databases. Grouping them based on the expression level of NET-related genes, we assessed the correlation between immune cell types and the risk score for having a poor prognosis of cALL, with an evaluation of the sensitivity of drugs used in cALL. We further divided the groups, integrating survival data. Subsequently, methods including multivariable Cox algorithms, least absolute shrinkage and selection operator (LASSO), and univariable were utilized to create a risk model predicting prognosis. Experiments in cell lines and animals were performed to explore the functions of TRIM8, a gene selected by the model. To validate the role of TRIM8 in leukemia development, lentivirus-mediated overexpression or knockdown of TRIM8 was employed in mice with T-ALL and B-ALL. Results: Kaplan-Meier (KM) analysis underscored the importance of differentially expressed genes identified in the groups divided by genes participated in NETs, with enrichment analysis showing the mechanism. Correlation analysis revealed significant associations with B cells, NK cells, mast cells, T cells, plasma cells, dendritic cells, and monocytes. The IC50 values of drugs such as all-trans-retinoic acid (ATRA), axitinib, doxorubicin, methotrexate, sorafenib, and vinblastine were increased, while dasatinib exhibited a lower IC50. A total of 13 NET-related genes were selected in constructing the risk model. In the training, testing, and merged cohorts, KM analysis demonstrated significantly improved survival for low-risk cALL patients compared to high-risk cALL patients (p < 0.001). The area under the curve (AUC) indicated strong predictive performance. Experiments in Jurkat and SUP-B15 revealed that TRIM8 knockdown decreased the proliferation of leukemia cell lines. Further experiments demonstrated a more favorable prognosis in mice with TRIM8-knockdown leukemia cells. Results of cell lines and animals showed better outcomes in prognosis when TRIM8 was knocked down. Conclusion: We identified a novelty in a prognostic model that could aid in the development of personalized treatments for cALL patients. Furthermore, it revealed that the expression of TRIM8 is a contributing factor to the proliferation of leukemia cells and worsens the prognosis of cALL.

Targeting neutrophils extracellular traps, a promising anti-thrombotic therapy for natural products from traditional Chinese herbal medicine.

Thrombi are the main cause of vascular occlusion and contribute significantly to cardiovascular events and death. Neutrophils extracellular traps (NETs)-induced thrombosis plays a vital role in thrombotic complications and it takes the main responsibility for the resistance of fibrinolysis. However, the conventional anti-thrombotic therapies are inadequate to treat NETs-induced thrombotic complications but carry a high risk of bleeding. Consequently, increased attention has shifted towards exploring novel anti-thrombotic treatments targeting NETs. Interestingly, accumulating evidences prove that natural products from traditional Chinese herbal medicines have a great potential to mitigate thrombosis through inhibiting generous NETs formation and degrading excessive NETs. In this review, we elaborated the formation and degradation of NETs and highlighted its pivotal role in immunothrombosis through interactions with platelets and coagulation factors. Since available anti-thrombotic drugs targeting NETs are deficient, we further summarized the natural products and compounds from traditional Chinese herbal medicines which exert effective actions on regulating NETs formation and also have anti-thrombotic effects. Our findings underscore the diverse effects of natural products in targeting NETs, including relieving inflammation and oxidative stress of neutrophils, inhibiting neutrophils activation and DNA efflux, suppressing granule proteins release, reducing histones and promoting DNA degradation. This review aims to highlight the significance of natural medicines in anti-thrombotic therapies through targeting NETs and to lay a groundwork for developing novel anti-thrombotic agents from traditional Chinese herbal medicines.

Antibodies and complement are key drivers of thrombosis.

Venous thromboembolism (VTE) is a common, deadly disease with an increasing incidence despite preventive efforts. Clinical observations have associated elevated antibody concentrations or antibody-based therapies with thrombotic events. However, how antibodies contribute to thrombosis is unknown. Here, we show that reduced blood flow enabled immunoglobulin M (IgM) to bind to FcµR and the polymeric immunoglobulin receptor (pIgR), initiating endothelial activation and platelet recruitment. Subsequently, the procoagulant surface of activated platelets accommodated antigen- and FcγR-independent IgG deposition. This leads to classical complement activation, setting in motion a prothrombotic vicious circle. Key elements of this mechanism were present in humans in the setting of venous stasis as well as in the dysregulated immunothrombosis of COVID-19. This antibody-driven thrombosis can be prevented by pharmacologically targeting complement. Hence, our results uncover antibodies as previously unrecognized central regulators of thrombosis. These findings carry relevance for therapeutic application of antibodies and open innovative avenues to target thrombosis without compromising hemostasis.

A biomimic anti-neuroinflammatory nanoplatform for active neutrophil extracellular traps targeting and spinal cord injury therapy.

Traumatic spinal cord injury (SCI) always leads to severe neurological deficits and permanent damage. Neuroinflammation is a vital process of SCI and have become a promising target for SCI treatment. However, the neuroinflammation-targeted therapy would hinder the functional recovery of spinal cord and lead to the treatment failure. Herein, a biomimic anti-neuroinflammatory nanoplatform (DHCNPs) was developed for active neutrophil extracellular traps (NETs) targeting and SCI treatment. The curcumin-loaded liposome with the anti-inflammatory property acted as the core of the DHCNPs. Platelet membrane and neutrophil membrane were fused to form the biomimic hybrid membrane of the DHCNPs for hijacking neutrophils and neutralizing the elevated neutrophil-related proinflammatory cytokines, respectively. DNAse I modification on the hybrid membrane could achieve NETs degradation, blood spinal cord barrier, and neuron repair. Further studies proved that the DHCNPs could reprogram the multifaceted neuroinflammation and reverse the SCI process via nuclear factor kappa-B (NF-κB) pathway. We believe that the current study provides a new perspective for neuroinflammation inhibition and may shed new light on the treatment of SCI.

Rapamycin and Hyperoside-Co-loaded Macrophage Delivery System Enhanced Pulmonary Fibrosis Therapy by Autophagy Upregulation and Epithelial-to-Mesenchymal Transition Inhibition.

Pulmonary fibrosis is a lethal interstitial lung disease, for which current treatments are inadequate in halting its progression. A significant factor contributing to the development of fibrosis is insufficient autophagy, which leads to increased fibroblast proliferation and collagen deposition. However, treatments aimed at upregulating autophagy often cause further lung pathology due to the disruption of epithelial cell balance. In response, we have developed a novel macrophage delivery system loaded with an epithelial-to-mesenchymal transition inhibitor, hyperoside (HYP), and an autophagy inducer, rapamycin (RAP). This system targets the fibrotic areas of the lung through chemotaxis, releases liposomes via macrophage extracellular traps, and effectively inhibits fibroblast proliferation while restoring the alveolar structure through the combined effects of RAP and HYP, ultimately reducing lung pathology without causing systemic toxicity. Our findings not only highlight a promising method to enhance autophagy-based treatments for pulmonary fibrosis but also demonstrate the potential of macrophages as effective nanocarriers for drug delivery.

Bone morphogenetic protein 4 inhibits corneal neovascularization by blocking NETs-induced disruption to corneal epithelial barrier.

Corneal neovascularization (CoNV) is the second leading cause of visual impairment worldwide, and current drugs have certain limitations. Inflammatory response is the core pathological process of CoNV. Neutrophil extracellular traps (NETs) are generated after neutrophil activation, which promotes neovascularization. Prior studies demonstrated that bone morphogenetic protein 4 (BMP4) could significantly reduce inflammation and CoNV formation, its exact molecular mechanism remains unclear. Therefore, we stimulated human peripheral blood neutrophils with phorbol myristate acetate (PMA) or deoxyribonuclease I (DNase I) to induce or inhibit NETs formation. By using corneal sutures and subconjunctival injections of NETs or DNase I, rat CoNV models were established. Compared with the suture group, NETs formation and inflammatory cell infiltration in the corneal stroma were significantly increased, corneal edema was aggravated, and the length, area and diameter of CoNV were significantly enhanced in the NETs group. Furthermore, by curetting the corneal epithelial apical junctional complexes (AJCs), a crucial component in preserving the function of the corneal epithelial barrier, we discovered that the damage of AJCs had a significant role in inducing CoNV formation. NETs could induce CoNV formation by injuring corneal epithelial AJCs. Finally, by comparing the aforementioned indicators after the intervention of BMP4, BMP4 inhibitor Noggin and NADPH oxidase (NOX) inhibitor, we finally demonstrated that BMP4 could inhibit NETs-induced inflammation and corneal epithelial AJC injury, repair corneal epithelial barrier function and eventually inhibit CoNV formation by blocking NOX-2-dependent NETs formation.

Identification of a biomarker to predict doxorubicin/cisplatin chemotherapy efficacy in osteosarcoma patients using primary, recurrent and metastatic specimens.

BACKGROUND: Doxorubicin and cisplatin are both first-line chemotherapeutics for osteosarcoma (OS) treatment. However, the efficacy of doxorubicin/cisplatin chemotherapy varies considerably. Thus, identifying an efficient diagnostic biomarker to distinguish patients with good and poor responses to doxorubicin/cisplatin chemotherapy is of paramount importance. METHODS: To predict the efficacy of doxorubicin/cisplatin chemotherapy, we analyzed the differentially expressed proteins in 37 resected OS samples, which were categorized into the primary group (PG), the recurrent group (RG) and the metastatic group (MG). The characteristics of the enriched differentially expressed proteins were assessed via GO and KEGG analyses. Protein‒protein interactions were identified to determine the relationships among the differentially expressed proteins. Receiver operating characteristic (ROC) curve analyses were performed to explore the clinical significance of the differentially expressed proteins. Parallel reaction monitoring (PRM) was used to validate the candidate proteins. Immunohistochemical (IHC) staining was performed to confirm the expression of cathepsin (CTSG) in patients with good and poor response to doxorubicin/cisplatin. RESULTS: A total of 9458 proteins were identified and quantified, among which 143 and 208 exhibited significant changes (|log2FC|>1, p < 0.05) in the RG and MG compared with the PG, respectively. GO and KEGG enrichment led to the identification of neutrophil extracellular traps (NETs). ROC curve analyses revealed 74 and 86 proteins with areas under the curve greater than 0.7 in the RG and MG, respectively. PRM validation revealed the statistical significance of CTSG, which is involved in NET formation, at the protein level in both the RG and MG. IHC staining of another cohort revealed that CTSG was prominently upregulated in the poor response group after treatment with doxorubicin/cisplatin. CONCLUSION: CTSG and its associated NETs are potential biomarkers with which the efficacy of doxorubicin/cisplatin chemotherapy could be predicted in OS patients.

Neutrophil extracellular traps: Potential targets for the treatment of rheumatoid arthritis with traditional Chinese medicine and natural products.

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease. Abnormal formation of neutrophil extracellular traps (NETs) at the synovial membrane leads to the release of many inflammatory cytokines, including IL-1ß, IL-6, and TNF-α. Elastase, histone H3, and myeloperoxidase, which are carried by NETs, damage the soft tissues of the joints and aggravate the progression of RA. The balance of NET formation coordinates the pro-inflammatory and anti-inflammatory effects and plays a key role in the development of RA. Therefore, when NETs are used as effector targets, highly targeted drugs with fewer side effects can be developed to treat RA without damaging the host immune system. Currently, an increasing number of studies have shown that traditional Chinese medicines and natural products can regulate the formation of NETs through multiple pathways to counteract RA, which shows great potential for the treatment of RA and has a promising future for clinical application. In this article, we review the latest biological progress in understanding NET formation, the mechanism of NETs in RA, and the potential targets or pathways related to the modulation of NET formation by Chinese medicines and natural products. This review provides a relevant basis for the use of Chinese medicines and natural products as natural adjuvants in the treatment of RA.

Metabolic dysregulation-triggered neutrophil extracellular traps exacerbate acute liver failure.

Acute liver failure (ALF) is an acute liver disease with a high mortality rate in clinical practice, characterized histologically by extensive hepatocellular necrosis and massive neutrophil infiltration. However, the role of these abnormally infiltrating neutrophils during ALF development is unclear. Here, in an ALF mouse model, metabolites were identified that promote the formation of neutrophil extracellular traps (NETs) in the liver, subsequently influencing macrophage differentiation and disease progression. ALF occurs with abnormalities in hepatic and intestinal metabolites. Abnormal metabolites (LTD4 and glutathione) can directly, or indirectly via reactive oxygen species, promote NET formation of infiltrating neutrophils, which subsequently regulate macrophages in a pro-inflammatory M1-like state, inducing an amplification of the destructive effects of inflammation. Together, this study provides new insights into the role of NETs in the pathogenesis of ALF.

The role of neutrophils in autoimmune diseases.

Historically, neutrophils have been primarily regarded as short-lived immune cells that act as initial responders to antibacterial immunity by swiftly neutralizing pathogens and facilitating the activation of adaptive immunity. However, recent evidence indicates that their roles are considerably more complex than previously recognized. Neutrophils comprise distinct subpopulations and can interact with various immune cells, release granular proteins, and form neutrophil extracellular traps. These functions are increasingly recognized as contributing factors to tissue damage in autoimmune diseases. This review comprehensively examines the physiological functions and heterogeneity of neutrophils, their interactions with other immune cells, and their significance in autoimmune diseases, including systemic lupus erythematosus, rheumatoid arthritis, antiphospholipid syndrome, antineutrophil cytoplasmic antibody-associated vasculitis, multiple sclerosis, and others. This review aims to provide a deeper understanding of the function of neutrophils in the development and progression of autoimmune disorders.

The pivotal role of neutrophil extracellular traps in cardiovascular diseases: Mechanisms and therapeutic implications.

Cardiovascular diseases (CVDs) continue to pose a significant burden on global health, prominently contributing to morbidity and mortality rates worldwide. Recent years have witnessed an increasing recognition of the intricate involvement of neutrophil extracellular traps (NETs) in the pathology of diverse cardiovascular conditions. This review provides a comprehensive analysis of the multifaceted functions of NETs in cardiovascular diseases, shedding light on the impact on atherosclerosis, myocardial infarction, heart failure, myocarditis, atrial fibrillation, aortic stenosis, and the potential therapeutic avenues targeting NETs.

Hypochlorous Acid-Modified Serum Albumin Causes NETosis in the Whole Blood Ex Vivo and in Isolated Neutrophils.

Type 2 diabetes mellitus (T2DM) is accompanied by halogenative stress resulting from the excessive activation of neutrophils and neutrophilic myeloperoxidase (MPO) generating highly reactive hypochlorous acid (HOCl). HOCl in blood plasma modifies serum albumin (Cl-HSA). We studied the formation of neutrophil extracellular traps (NETs) in the whole blood and by isolated neutrophils under the action of Cl-HSA. It was found that Cl-HSA induces neutrophil priming and NETosis. MPO-containing as well as MPO-free NETs were found. These NETs with different composition can be a product of NETosis of one and the same neutrophil. NET formation in neutrophils with vacuolated cytoplasm was detected. In the presence of Cl-HSA, acceleration of NET degradation was observed. Accelerated NET degradation and neutrophil priming can be the factors contributing to the development of complications in T2DM.

Timing, initiation and function: An in-depth exploration of the interaction network among neutrophil extracellular traps related genes in acute pancreatitis.

BACKGROUND: Exogenous inhibition of neutrophil extracellular traps (NETs) was believed to alleviate acute pancreatitis (AP). This study aimed to comprehensively explore the key biological behavior of NETs including timing and pathogenesis in AP by integrating of single cell RNA sequencing(scRNA-seq) and bulk RNA-seq. METHODS: Differentially expressed NETs-related genes and the hub genes of NETs were screened by bulk RNA-seq. ScRNA-seq was used to identify the cell types in pancreas of AP mice and to depict the transcriptomic maps in neutrophils. The mouse AP models were build to verify the timing of initiation of NETs and underlying pathogenesis of damage on pancreas acinar cells. RESULTS: Tlr4 and Ccl3 were screened for hub genes by bulk RNA-seq. The trajectory analysis of neutrophils showed that high expression of Ccl3, Cybb and Padi4 can be observed in the middle stage during AP. Macrophages might be essential in the biological behavior of neutrophils and NETs. Through animal models, we presented that extensive NETs structures were formed at mid-stage of inflammation, accompanied by more serious pancreas and lung damage. NETs might promote necroptosis and macrophage infiltration in AP, and the damage on pancreatic injury could be regulated by Tlr4 pathway. Ccl3 was considered to recruit neutrophils and promote NETs formation. CONCLUSION: The findings explored the underlying timing and pathogenesis of NETs in AP for the first time, which provided gene targets for further studies.

Zinc Supplementation Reduces the Formation of Neutrophil Extracellular Traps by Decreasing the Expression of Peptidyl Arginine Deiminase 4.

SCOPE: Neutrophils play a decisive role during the immediate defense against infections. However, as observed during rheumatoid arthritis, activated neutrophils can also cause tissue damage. Previous studies indicate that zinc supplementation may alter certain neutrophil functions. However, precise underlying mechanisms and possible effects of zinc deficiency remain incompletely understood. The objective of this study is to investigate the effects of changes in zinc status on formation of neutrophil extracellular traps (NETs) and other fundamental neutrophil functions. METHODS AND RESULTS: Interleukin (IL)-17 and tumor necrosis factor (TNF)-α are used to simulate the inflammatory environment observed in autoimmune diseases. The study analyzes the impact of the zinc status on NETs release, using a fluorescence plate reader, and on the expression of peptidylarginine deiminase 4 (PAD4), S100A8/A9, and certain cytokines by PCR and western blot. These results show that zinc supplementation significantly reduces NETs formation and downregulates PAD4 protein expression. Zinc supplementation results in increased protein expression of interleukin-1 receptor antagonist (IL-1RA) and IL-8 in stimulated cells. CONCLUSION: The results suggest that changes in extracellular zinc availability may influence the functions of neutrophils. Therefore, maintaining an appropriate zinc level is advisable for preserving innate immunity and to prevent hyper-activation of neutrophils.