Although studies have evaluated the presence of cell-free DNA and neutrophil extracellular traps (NETs) in acute respiratory distress syndrome (ARDS), the kinetics of NET formation during the early ICU admission and whether plasma NET markers correlate with clinical outcomes in patients with moderate-to-severe hypoxemia remain unknown. We sought to determine whether serial plasma NET marker levels in study participants collected over 48 h post enrollment predict disease severity and mortality in non-COVID-19 ARDS patients.

We obtained previously collected plasma samples (trial enrollment, 24 h, 48 h) from 200 randomly selected ARDS participants in the completed Reevaluation of Systemic Early Neuromuscular Blockade (ROSE) Trial, as well as from 20 healthy control donors. We determined plasma levels of surrogate biomarkers for NETs using a cell-free DNA fluorescence assay and a plasma myeloperoxidase (MPO)-DNA complex ELISA. We correlated these surrogate biomarker levels with clinical outcomes from the ROSE trial study participants.

ROSE plasma samples demonstrated significantly higher NET levels compared to healthy donor controls. Individual study participant NET levels did not change over the forty-eight hours after trial enrollment. Higher levels of both surrogate markers correlated with fewer ventilator-free days, but only cell free-DNA correlated with mortality and higher illness severity scores.

Surrogate markers for plasma NET levels measured in patients with moderate or severe ARDS correlate directly with adverse clinical outcomes and may serve as biomarkers for predicting severe disease. Further studies of surrogate biomarkers for NET formation in moderate-to-severe ARDS are warranted.

Our previous studies have demonstrated that neutrophils play a key role in septic organ injury partly through the excessive formation of neutrophil extracellular traps (NETs) and that exosomes participate in the regulation of NET formation during sepsis. Therefore, this study aimed to determine whether neutrophil-derived exosomes promote the formation of NETs and induce multiple organ dysfunction during sepsis. Initially, polymorphonuclear neutrophil (PMN)-derived exosomes following in vitro stimulation with PBS or LPS (1 μg/mL) for 6 h. In vivo, PMN-derived exosomes were intravenously administered to wild-type C57BL/6 mice. Then, histopathological injury and NET formation in multiple organs were evaluated. In vitro, PMN-derived exosomes were cocultured with PMNs freshly isolated from healthy volunteers, and subsequently, NET formation and activation of associated molecular pathways were detected. Administration of LPS-stimulated PMN-derived exosomes in mice significantly enhanced NET formation, resulting in multi-organ inflammation and tissue injury. In vitro coculture experiments also demonstrated that exosomes from LPS-stimulated PMNs promote ROS-dependent NET formation. Proteomic analysis revealed enrichment of matrix metalloproteinase 9 (MMP9) expression in exosomes from LPS-stimulated PMNs, and further mechanistic investigations showed that exosomal MMP9 induced NET formation through the p38 MAPK pathway. Clinical data analysis suggests a close association between sepsis severity/prognosis and plasma-derived exosomal MMP9 expression levels. PMN-derived exosomes facilitate the excessive formation of NETs in sepsis, leading to the subsequent development of multiple organ dysfunction. This discovery reveals a novel role for PMN-derived exosomes in the pathogenesis of sepsis-related multiple organ dysfunction and suggests their potential as prognostic indicators for this condition.

Hemostatic imbalance in dogs with sepsis is characterized by hypercoagulability and hypofibrinolysis. We aimed to determine whether these abnormalities are unique features of sepsis or are also present in dogs with non-septic critical illness. Secondary aims were to assess relationships between coagulation assay results and circulating markers of neutrophil extracellular traps (NETs), and to relate coagulation assay abnormalities with survival in dogs with sepsis.

This prospective single-center observational cohort study enrolled 55 client-owned dogs that satisfied at least 2 systemic inflammatory response syndrome (SIRS) criteria. Dogs with a bacterial infection were categorized as sepsis, those without evidence of infection were categorized as non-infectious systemic inflammation (nSIRS). Clotting times, fibrinogen and D-dimer concentrations, and activities of antithrombin (AT), antiplasmin (AP), thrombin activatable fibrinolysis inhibitor (TAFI), and total and active plasminogen activator inhibitor-1 (PAI-1) were measured. Thrombin generation and overall hemostasis potential assays were performed and concentrations of cell-free DNA (cfDNA) and H3.1 nucleosomes quantitated.

Compared to dogs with nSIRS, dogs with sepsis had higher fibrinogen concentrations, greater endogenous thrombin potential, higher AP and TAFI activities and greater overall hemostasis and coagulation potential values. H3.1 nucleosome and cfDNA concentrations were strongly correlated and significantly associated with various coagulation variables. In dogs with sepsis, non-survivors had lower AT activity, and higher active PAI-1 and H3.1 nucleosome concentrations.

Relative to non-septic critically ill dogs, dogs with sepsis are hyperfibrinogenemic, hypercoagulable and have higher AP and TAFI activities. Concentrations of H3.1 nucleosomes and active PAI-1 and AT activity might have prognostic value in dogs with sepsis.

Neutrophils play a key role in sepsis-associated acute kidney injury (SAKI), a common and life-threatening complication of organ failure. High mobility group box 1 (HMGB1) modulates inflammatory responses and the formation of neutrophil extracellular traps (NETs). The present work aimed to explore whether HMGB1 lactylation promotes NET formation and exacerbates SAKI.

Venous blood samples were collected from healthy volunteers and SAKI patients. A SAKI mouse model was established using the cecal ligation and puncture method. A coculture system of macrophage-derived exosomes and neutrophils was established. Macrophage-derived exosomes were isolated and identified. ELISAs, immunofluorescence staining, coimmunoprecipitation, and Western blotting were utilized to determine protein levels.

Elevated blood lactate levels were associated with increased HMGB1 levels in patients with SAKI. In mouse models, lactate increased HMGB1 expression, promoted NET formation, and exacerbated SAKI. Lactate stimulated M1 macrophages to secrete exosomes, leading to the accumulation and release of HMGB1 in the cytoplasm. Additionally, lactate promoted HMGB1 lactylation in macrophages, triggering the release of mitochondrial DNA from neutrophils and activating the cyclic GMP‒AMP synthase/stimulator of interferon genes pathway.

This study revealed that lactate-induced HMGB1 lactylation in macrophages plays a role in promoting NET formation in SAKI through the cGAS/STING pathway. These findings suggest that HMGB1 could be a potential target for therapeutic intervention in SAKI.

Neutrophils and neutrophil extracellular traps (NETs) have been identified as crucial contributors in several neuroinflammatory models, such as stroke and traumatic brain injury, but their role in sepsis-associated encephalopathy (SAE) has not been thoroughly investigated.

In this study, we established an SAE model using cecal ligation puncture (CLP) surgery to examine neutrophil infiltration and NETs formation. A protein arginine deiminase 4 (PAD4) inhibitor, GSK484, was employed to suppress NETs release. To assess changes in hippocampal gene expression induced by GSK484 treatment in CLP mice, we utilized RNA sequencing (RNA-Seq) combined with bioinformatics analysis. Additionally, the Elisa, cognitive function test, western bolt and immunofluorescence staining were used to measured hippocampal inflammatory cytokine, cognitive function, and the protein levels of tight junctions (TJs) and adherens junctions (AJs) in SAE mice. We also established a Transwell™ co-culture system using bEnd.3 cells and bone marrow-derived neutrophils to examine the effects of GSK484 on endothelial cell function. This comprehensive approach allowed us to evaluate the impact of NETs inhibition on neuroinflammation, cognitive function, and the underlying molecular mechanisms in the CLP-induced SAE model.

Our findings revealed that neutrophils were significantly overactivated, releasing abundant NETs in the hippocampus of CLP-induced SAE mice. Inhibition of NET formation using GSK484 led to reduced neuroinflammatory responses, improved blood-brain barrier (BBB) integrity, and enhanced survival rates and cognitive function in SAE mice. RNA-Seq and bioinformatics analyses identified the Wnt signaling pathway as the most significant pathway affected. Subsequent experiments demonstrated that NETs inhibition alleviated BBB damage primarily by increasing the expression of Occludin, a TJs protein, and promoting the formation of the VCL/β-catenin/VE-cadherin complex at AJs, mediated by the Wnt3/β-catenin/TCF4 signaling pathway.

Our results suggest that inhibition of NETs may protect BBB permeability and cognitive function through the Wnt3/β-catenin/TCF4 signaling pathway in the context of CLP-induced SAE, which provides a promising strategy for SAE therapy.

Iron metabolism of neutrophils plays a vital role in neutrophil extracellular trap (NET) formation, which presents as one of the major hurdles to the immune response in the tumor microenvironment. Here, we developed a peptide-drug conjugate (PDC)-based transformable iron nanochelator (TIN) equipped with the ability to regulate the iron metabolism of neutrophils, endowing inhibition of NET formation and the ensuing immunosuppression functions. The TIN could expose the iron-binding motifs through neutrophil elastase-mediated morphological transformation from nanoparticles to β-sheet nanofibers, which further evolve into stable α-helix nanofibers after chelation with iron(II) ions. This process enables a highly specific regulation of iron(II) ions of neutrophils, which turns into an efficient way of inhibiting NET formation and improving the immune response. Furthermore, the TIN showed an improved therapeutic effect in combination with protein arginine deiminase 4 inhibitors and synergistically boosted the anti-PD-L1 treatment. This study designates an iron-regulation strategy to inhibit NET formation, which provides an alternative approach to immune modulation from the perspective of targeting the iron metabolism of neutrophils in cancer immunotherapy.

Metabolic dysfunction-associated steatohepatitis (MASH) fibrosis is a reversible stage of liver disease accompanied by inflammatory cell infiltration. Neutrophils extrude a meshwork of chromatin fibers to establish neutrophil extracellular traps (NETs), which play important roles in inflammatory response regulation. Our previous work demonstrated that NETs promote HCC in MASH. However, it is still unknown if NETs play a role in the molecular mechanisms of liver fibrosis.

Following 12 weeks of Western diet/carbon tetrachloride, MASH fibrosis was identified in C57BL/6 mice with increased NET formation. However, NET depletion using DNase I treatment or mice knocked out for peptidyl arginine deaminase type IV significantly attenuated the development of MASH fibrosis. NETs were demonstrated to induce HSCs activation, proliferation, and migration through augmented mitochondrial and aerobic glycolysis to provide additional bioenergetic and biosynthetic supplies. Metabolomic analysis revealed markedly an altered metabolic profile upon NET stimulation of HSCs that were dependent on arachidonic acid metabolism. Mechanistically, NET stimulation of toll-like receptor 3 induced cyclooxygenase-2 activation and prostaglandin E2 production with subsequent HSC activation and liver fibrosis. Inhibiting cyclooxygenase-2 with celecoxib reduced fibrosis in our MASH model.

Our findings implicate NETs playing a critical role in the development of MASH hepatic fibrosis by inducing metabolic reprogramming of HSCs through the toll-like receptor 3/cyclooxygenase-2/cyclooxygenase-2 pathway. Therefore, NET inhibition may represent an attractive treatment target for MASH liver fibrosis.

Neutrophil granulocytes are important parts of the defence against bacterial infections. Their action is a two-edged sword, the mediators killing the intruding bacteria are at the same time causing tissue damage. Neutrophil activation is part of the dysregulated immune response to infection defining sepsis and neutrophil elastase is one of the powerful proteases causing both effects and damage. Inhibition of neutrophil elastase has been tried in sepsis and ARDS, so far with inconclusive results.

We used positron emission tomography (PET) combined with computed tomography (CT) and the selective and specific neutrophil elastase inhibitor PET-tracer [11C]GW457427 ([11C]NES), in an intensive care unit porcine Escherichia coli sepsis model with the primary aim to visualise the biodistribution of neutrophil elastase in the initial acute phase of the septic reaction. Repeated PET-CT investigations were performed before and after induction of sepsis.

At baseline [11C]NES uptake was found in the bone marrow, spleen and liver. The uptake in the bone marrow was markedly increased two hours into the sepsis, whereas in spleen and liver the uptake was not as markedly changed compared to baseline. At 4 h after the sepsis induction [11C]NES in the bone marrow decreased while the uptake increased in the spleen, liver and lungs.

The neutrophil elastase PET-tracer [11C]NES is a novel and unique instrument to study the acute innate neutrophil immune response in sepsis and associated vital organ failure. We here present images and quantitative data of the neutrophil elastase distribution the first hours of acute experimental sepsis. Surprisingly, a pronounced increase of neutrophil elastase was found in the bone marrow 2 h into the sepsis reaction followed at 4 h by increase in the liver, spleen and lungs and a concomitant reduction of the tracer uptake in bone marrow.

Neutrophils release neutrophil extracellular traps (NETs) as part of a healthy host immune response. NETs physically trap and kill pathogens as well as activating and facilitating crosstalk between immune cells and complement. Excessive or inadequately resolved NETs are implicated in the underlying pathophysiology of sepsis and other inflammatory diseases, including amplification of the inflammatory response and inducing thrombotic complications. Here, we review the growing evidence implicating neutrophils and NETs as central players in the dysregulated host immune response. We discuss potential strategies for modifying NETs to improve patient outcomes and the need for careful patient selection.

Nonhuman primate models that closely emulate the disease course, pathogenesis, and supportive care provided to human patients in the modern intensive care unit with bacterial sepsis are urgently needed to study pathogenesis and assess novel therapies. We therefore developed a non-human primate model of septic shock that includes supportive care akin to a modern intensive care unit. In this study, we characterized pathogen kinetics and evaluated the physiologic, immunologic, and pathologic responses in this model of septic shock induced by the clinically relevant pathogen Klebsiella pneumoniae across a three-log dose range. We observed dose-dependent bacteremia and circulating levels of Klebsiella pneumoniae DNA and endotoxin. Tachycardia and hypotension occurred in all animals and the study endpoint occurred in 8 of 12 animals that were euthanized. The infused bacterial dose was significantly associated with the severity of renal insufficiency and coagulopathy. Neutrophil activation evidenced by increased CD11b expression, decreased CD62L expression, and increased circulating levels of myeloperoxidase, lactoferrin, and neutrophil extracellular traps; monocyte activation evidenced by increased circulating levels of interleukin-6, tumor necrosis factor-alpha, granulocyte-macrophage colony-stimulating factor, and monocyte chemotactic protein-1; and endothelial activation evidenced by increased circulating levels of syndecan-1 and angiopoietin-II were all consistent with human sepsis. Our model provides an opportunity to study pathogenesis and investigate novel therapeutics for the treatment of bacterial sepsis in the setting of modern supportive care.IMPORTANCEThere is currently a disconnect between the efficacy of sepsis therapies in pre-clinical animal models and human clinical trials. Therefore, developing nonhuman primate models that closely mimic human sepsis pathogenesis to study novel host-targeted therapeutics is a priority. In this study, we developed a model of septic shock with a clinically relevant bacteria (Klebsiella pneumoniae) that provides standard supportive care including mechanical ventilation, invasive hemodynamic monitoring, volume resuscitation, vasopressors, antibiotics, and steroids. In a dose-dependent manner, we observed that this model closely emulates the hemodynamic, end-organ dysfunction, and cellular and soluble responses associated with human sepsis. This validated model provides a unique opportunity to study the pathogenesis of acute septic shock and evaluate host-directed therapeutics in a large animal model that closely emulates the modern-day intensive care unit and supportive critical care.

Sepsis is characterized by a dysregulated immune response and is very difficult to treat. In the cecal ligation and puncture (CLP) mouse model, we show that nanomedicines can effectively alleviate systemic and local septic events by targeting neutrophils. Specifically, by decorating the surface of clinical-stage dexamethasone liposomes with cyclic arginine-glycine-aspartic acid (cRGD) peptides, we promote their engagement with neutrophils in the systemic circulation, leading to their prominent accumulation at primary and secondary sepsis sites. cRGD-targeted dexamethasone liposomes potently reduce immature circulating neutrophils and neutrophil extracellular traps in intestinal sepsis induction sites and the liver. Additionally, they mitigate inflammatory cytokines systemically and locally while preserving systemic IL-10 levels, contributing to lower IFN-γ/IL-10 ratios as compared to control liposomes and free dexamethasone. Our strategy addresses sepsis at the cellular level, illustrating the use of neutrophils both as a therapeutic target and as a chariot for drug delivery.

Sepsis is a severe disease that occurs when the body's immune system reacts excessively to infection. The body's response, which includes an intense antibacterial reaction, can damage its tissues and organs. Neutrophils are the major components of white blood cells in circulation, play a vital role in innate immunity while fighting against infections, and are considered a feature determining sepsis classification. There is a plethora of basic research detailing neutrophil functioning, among which, the study of neutrophil extracellular traps is providing novel insights into mechanisms and treatments of sepsis. This review explores their functions, dysfunctions, and influences in the context of sepsis. The interplay between neutrophils and the human microbiome and the impact of DNA methylation on neutrophil function in sepsis are crucial areas of study. The interaction between neutrophils and the human microbiome is complex, particularly in the context of sepsis, where dysbiosis may occur. We highlight the importance of deciphering neutrophils' functional alterations and their epigenetic features in sepsis because it is critical for defining sepsis endotypes and opening up the possibility for novel diagnostic methods and therapy. Specifically, epigenetic signatures are pivotal since they will provide a novel implication for a sepsis diagnostic method when used in combination with the cell-free DNA. Research is exploring how specific patterns of DNA methylation in neutrophils, detectable in cell-free DNA, could serve as biomarkers for the early detection of sepsis.

Background/Objectives: Neutrophils, as the first line of defense in the immune response, produce neutrophil extracellular traps (NETs) upon activation, which are significant in the pathogenesis and organ damage in sepsis. This study aims to explore the clinical value of myeloperoxidase-DNA (MPO-DNA) and cell-free DNA (cf-DNA) in sepsis patients. Methods: Clinical data were collected from 106 sepsis patients, 25 non-sepsis patients, and 51 healthy controls. Sequential Organ Failure Assessment (SOFA) scores were calculated, and levels of MPO-DNA) complexes and cf-DNA were measured using specific kits. Correlation analyses assessed relationships between indicators, while logistic regression identified independent risk factors. Receiver operating characteristic (ROC) curves calculated the area under the curve (AUC) to evaluate the diagnostic value of the biomarkers. Results: Sepsis patients exhibited significantly elevated levels of MPO-DNA and cf-DNA compared to non-sepsis patients and healthy controls. In sepsis patients, MPO-DNA and cf-DNA levels correlated with inflammation, coagulation, and organ damage indicators, as well as procalcitonin (PCT) levels and SOFA scores. Both C-reactive protein (CRP) and cf-DNA were identified as independent risk factors for sepsis, demonstrating moderate diagnostic value. ROC analysis showed that the combination of MPO-DNA and CRP (AUC: 0.837) enhances the AUC value of CRP (0.777). Conclusions: In summary, elevated serum levels of MPO-DNA and cf-DNA in sepsis patients correlate with SOFA scores and PCT levels, providing reference value for sepsis diagnosis in clinical settings.

Neutrophil extracellular traps (NETs), crucial in immune defense mechanisms, are renowned for their propensity to expel decondensed chromatin embedded with inflammatory proteins. Our comprehension of NETs in pathogen clearance, immune regulation and disease pathogenesis, has grown significantly in recent years. NETs are not only pivotal in the context of infections but also exhibit significant involvement in sterile inflammation. Evidence suggests that excessive accumulation of NETs can result in vessel occlusion, tissue damage, and prolonged inflammatory responses, thereby contributing to the progression and exacerbation of various pathological states. Nevertheless, NETs exhibit dual functionalities in certain pathological contexts. While NETs may act as autoantigens, aggregated NET complexes can function as inflammatory mediators by degrading proinflammatory cytokines and chemokines. The delineation of molecules and signaling pathways governing NET formation aids in refining our appreciation of NETs' role in immune homeostasis, inflammation, autoimmune diseases, metabolic dysregulation, and cancer. In this comprehensive review, we delve into the multifaceted roles of NETs in both homeostasis and disease, whilst discussing their potential as therapeutic targets. Our aim is to enhance the understanding of the intricate functions of NETs across the spectrum from physiology to pathology.

In end-stage heart failure, which is characterized by persistent or progressive ventricular dysfunction despite optimal medical therapy, a left ventricular assist device (LVAD) can be beneficial. Congestive heart failure provokes inflammatory and prothrombotic activation. The aim of this study was to evaluate the serum concentration of citrullinated histone 3 (CH3) representing neutrophil extracellular trap (NET) formation in patients referred for LVAD implantation. There were 10 patients with a median age of 61 (57-65) years enrolled in a prospective single-center analysis who underwent LVAD implantation. The CH3 plasma concentration was measured preoperatively and on the 1st and 7th postoperative days, followed by control measurements on the median (Q1-3) 88th (49-143) day. The preoperative CH3 concentration strongly correlated with brain natriuretic peptide (r = 0.879, p < 0.001). Significant differences in CH3 serum concentration were observed between pre- and postoperative measurements, including an increase on the first postoperative day (p < 0.001), as well as a decrease on the seventh day (p = 0.016) and in follow-up (p < 0.001). CH3 concentration, as a marker of NET formation, decreases after LVAD implantation.

Modulating SYK has been demonstrated to have impacts on pathogenic neutrophil responses in COVID-19. During sepsis, neutrophils are vital in early bacterial clearance but also contribute to the dysregulated immune response and organ injury when hyperactivated. Here, we evaluated the impact of R406, the active metabolite of fostamatinib, on neutrophils stimulated by LPS. We demonstrate that R406 was able to effectively inhibit NETosis, degranulation, ROS generation, neutrophil adhesion, and the formation of CD16low neutrophils that have been linked to detrimental outcomes in severe sepsis. Further, the neutrophils remain metabolically active, capable of releasing cytokines, perform phagocytosis, and migrate in response to IL-8. Taken together, this data provides evidence of the potential efficacy of utilizing fostamatinib in bacterial sepsis.

Sepsis remains a major cause of mortality in intensive care units (ICUs). Prompt diagnosis and effective management are imperative for better outcomes. In this systematic review and meta-analysis, we explore the potential of circulating cell-free DNA (cfDNA), as a promising tool for early sepsis detection and prognosis assessment, aiming to address limitations associated with traditional diagnostic methods.

Following PRISMA guidelines, we collected relevant literature from thirteen databases. Studies were included if they analyzed quantitative diagnostic or prognostic cfDNA levels in humans in case of sepsis. We collected data on basic study characteristics, baseline patient demographics (e.g. age and sex), and cfDNA levels across different stages of sepsis. Pooled SMD with 95%-CI was calculated, and Comprehensive Meta-Analysis (CMA) software facilitated meta-analysis. Receiver operating characteristic (ROC) curves were generated to assess cfDNA's combined sensitivity and specificity in diagnostics and prognostics.

We included a final of 44 studies, of which, only 32 with 2950 participants were included in the meta-analysis. cfDNA levels were higher in septic patients compared to healthy controls (SMD = 3.303; 95%-CI [2.461-4.145], p<0.01). Furthermore, cfDNA levels were higher in non-survivors than survivors (SMD = 1.554; 95%-CI [0.905-2.202], p<0.01). Prognostic studies demonstrated a pooled sensitivity and specificity of 0.78, while diagnostic studies showed a sensitivity of 0.81 and a specificity of 0.87.

These findings show that cfDNA levels are significantly higher in sepsis patients compared to control groups and non-survivors in comparison to survivors among both adult and pediatric populations.

Sepsis is a systemic inflammatory response syndrome caused by the invasion of pathogenic microorganisms. Despite major advances in diagnosis and technology, morbidity and mortality remain high. The level of neutrophil extracellular traps (NETs) is closely associated with the progression and prognosis of sepsis, suggesting the regulation of NET formation as a new strategy in sepsis treatment. Owing to its pleiotropic effects, atorvastatin, a clinical lipid-lowering drug, affects various aspects of sepsis-related inflammation and immune responses. To align closely with clinical practice, we combined it with imipenem for the treatment of sepsis. In this study, we used a cecum ligation and puncture-induced lung injury mouse model and employed techniques including western blot, immunofluorescence, and enzyme-linked immunosorbent assay to measure the levels of NETs and other sepsis-related lung injury indicators. Our findings indicate that atorvastatin effectively inhibited the formation of NETs. When combined with imipenem, it significantly alleviated lung injury, reduced systemic inflammation, and improved the 7-day survival rate of septic mice. Additionally, we explored the inhibitory mechanism of atorvastatin on NET formation in vitro, revealing its potential action through the ERK/NOX2 pathway. Therefore, atorvastatin is a potential immunomodulatory agent that may offer new treatment strategies for patients with sepsis in clinical settings.

Neutrophil extracellular traps (NETs) seriously impede diabetic wound healing. The disruption or scavenging of NETs using deoxyribonuclease (DNase) or cationic nanoparticles has been limited by liberating trapped bacteria, short half-life, or potential cytotoxicity. In this study, a positive correlation between the NETs level in diabetic wound exudation and the severity of wound inflammation in diabetic patients is established. Novel NETs scavenging bio-based hydrogel microspheres 'micro-cage', termed mPDA-PEI@GelMA, is engineered by integrating methylacrylyl gelatin (GelMA) hydrogel microspheres with cationic polyethyleneimine (PEI)-functionalized mesoporous polydopamine (mPDA). This unique 'micro-cage' construct is designed to non-contact scavenge of NETs between nanoparticles and the diabetic wound surface, minimizing biological toxicity and ensuring high biosafety. NETs are introduced into 'micro-cage' along with wound exudation, and cationic mPDA-PEI immobilizes them inside the 'micro-cage' through a strong binding affinity to the cfDNA web structure. The findings demonstrate that mPDA-PEI@GelMA effectively mitigates pro-inflammatory responses associated with diabetic wounds by scavenging NETs both in vivo and in vitro. This work introduces a novel nanoparticle non-contact NETs scavenging strategy to enhance diabetic wound healing processes, with potential benefits in clinical applications.

Neutrophils perform essential functions in antimicrobial defense and tissue maintenance at mucosal barriers. However, a dysregulated neutrophil response and, in particular, the excessive release of neutrophil extracellular traps (NETs) are implicated in the pathology of various diseases. In this review, we provide an overview of the basic concepts related to neutrophil functions, including NET formation, and discuss the mechanisms associated with NET activation and function in the context of the prevalent oral disease periodontitis.

A rapid, quantitative serum S100A8/A9 (calprotectin) lateral flow test in combination with clinical status predicted outcomes in people hospitalised with COVID-19 and associated with a patient cluster driven by markers of neutrophil activation https://bit.ly/48e1BIv.

Apart from being a significant line of defense in the host defense system, neutrophils have many immunological functions. Although there are not many publications that accurately present the functions of neutrophils in relation to oncological pathology, their activity and implications have been studied a lot recently. This review aims to extensively describe neutrophils functions'; their clinical implications, especially in tumor pathology; the value of clinical markers related to neutrophils; and the implications of neutrophils in onco-anesthesia. This review also aims to describe current evidence on the influence of anesthetic drugs on neutrophils' functions and their potential influence on perioperative outcomes.

Neutrophil extracellular traps (NETs) are extracellular fibers composed of deoxyribonucleic acid (DNA) and decorated proteins produced by neutrophils. Recently, NETs have been associated with the development of many diseases, including tumors. Herein, we reviewed the correlation between NETs and tumors. In addition, we detailed active compounds from traditional herbal medicine formulations that inhibit NETs, related nanodrug delivery systems, and antibodies that serve as "guiding moieties" to ensure targeted delivery to NETs. Furthermore, we discussed the strategies used by pathogenic microorganisms to evade NETs.

Sepsis is a clinical syndrome defined as life-threatening organ dysfunction caused by a dysregulated host response to infection. Sepsis is a highly heterogeneous syndrome with distinct phenotypes that impact immune function and response to infection. To develop targeted therapeutics, immunophenotyping is needed to identify distinct functional phenotypes of immune cells. In this study, we utilized our Organ-on-Chip assay to categorize sepsis patients into distinct phenotypes using patient data, neutrophil functional analysis, and proteomics.

Following informed consent, neutrophils and plasma were isolated from sepsis patients in the Temple University Hospital ICU (n=45) and healthy control donors (n=7). Human lung microvascular endothelial cells (HLMVEC) were cultured in the Organ-on-Chip and treated with buffer or cytomix ((TNF/IL-1β/IFNγ). Neutrophil adhesion and migration across HLMVEC in the Organ-on-Chip were used to categorize functional neutrophil phenotypes. Quantitative label-free global proteomics was performed on neutrophils to identify differentially expressed proteins. Plasma levels of sepsis biomarkers and neutrophil extracellular traps (NETs) were determined by ELISA.

We identified three functional phenotypes in critically ill ICU sepsis patients based on ex vivo neutrophil adhesion and migration patterns. The phenotypes were classified as: Hyperimmune characterized by enhanced neutrophil adhesion and migration, Hypoimmune that was unresponsive to stimulation, and Hybrid with increased adhesion but blunted migration. These functional phenotypes were associated with distinct proteomic signatures and differentiated sepsis patients by important clinical parameters related to disease severity. The Hyperimmune group demonstrated higher oxygen requirements, increased mechanical ventilation, and longer ICU length of stay compared to the Hypoimmune and Hybrid groups. Patients with the Hyperimmune neutrophil phenotype had significantly increased circulating neutrophils and elevated plasma levels NETs.

Neutrophils and NETs play a critical role in vascular barrier dysfunction in sepsis and elevated NETs may be a key biomarker identifying the Hyperimmune group. Our results establish significant associations between specific neutrophil functional phenotypes and disease severity and identify important functional parameters in sepsis pathophysiology that may provide a new approach to classify sepsis patients for specific therapeutic interventions.

Plasma cell-free DNA (cfDNA), a marker of disease severity in sepsis, is a recognized driver of thromboinflammation and a potential therapeutic target. In sepsis, plasma cfDNA is mostly derived from neutrophil extracellular trap (NET) degradation. Proposed NET-directed therapeutic strategies include preventing NET formation or accelerating NET degradation. However, NET digestion liberates pathogens and releases cfDNA that promote thrombosis and endothelial cell injury. We propose an alternative strategy of cfDNA and NET stabilization with chemokine platelet factor 4 (PF4, CXCL4). We previously showed that human PF4 (hPF4) enhances NET-mediated microbial entrapment. We now show that hPF4 interferes with thrombogenicity of cfDNA and NETs by preventing their cleavage to short-fragment and single-stranded cfDNA that more effectively activates the contact pathway of coagulation. In vitro, hPF4 also inhibits cfDNA-induced endothelial tissue factor surface expression and von Willebrand factor release. In vivo, hPF4 expression reduced plasma thrombin-antithrombin (TAT) levels in animals infused with exogenous cfDNA. Following lipopolysaccharide challenge, Cxcl4-/- mice had significant elevation in plasma TAT, cfDNA, and cystatin C levels, effects prevented by hPF4 infusion. These results show that hPF4 interacts with cfDNA and NETs to limit thrombosis and endothelial injury, an observation of potential clinical benefit in the treatment of sepsis.

Sepsis is a systemic inflammatory disease caused by severe infections that involves multiple systemic organs, among which the lung is the most susceptible, leaving patients highly vulnerable to acute lung injury (ALI). Refractory hypoxemia and respiratory distress are classic clinical symptoms of ALI caused by sepsis, which has a mortality rate of 40%. Despite the extensive research on the mechanisms of ALI caused by sepsis, the exact pathological process is not fully understood. This article reviews the research advances in the pathogenesis of ALI caused by sepsis by focusing on the treatment regimens adopted in clinical practice for the corresponding molecular mechanisms. This review can not only contribute to theories on the pathogenesis of ALI caused by sepsis, but also recommend new treatment strategies for related injuries.

Although cell-free DNA (cfDNA) is an emerging sepsis biomarker, the use of cfDNA, especially as diagnostic and prognostic indicators, has surprisingly not been systemically analyzed. Data of adult patients with sepsis that conducted cfDNA measurement within 24 h of the admission was collected from PubMed, ScienceDirect, Scopus, and Cochrane Library until October 2022. The Quality in Prognosis Studies (QUIPS) and Quality Assessment in Diagnostic Studies-2 (QUADAS-2) tools were used to reduce the risk of biased assessment. The mean difference (MD) of cfDNA concentration and the standardized mean difference (SMD) between populations was calculated using Review Manager (RevMan) version 5.4.1 package software. Pooled analysis from 18 included studies demonstrated increased serum cfDNA levels in sepsis when compared with healthy control (SMD = 1.02; 95% confidence interval (CI) 0.46-1.57) or non-sepsis patients in the intensive care unit (ICU) (SMD = 1.03; 95% CI 0.65-1.40), respectively. Meanwhile, a slight decrease in the statistical value was observed when compared with non-sepsis ICU patients with SIRS (SMD = 0.74; 95% 0.41-1.06). The lower cfDNA levels were also observed in sepsis survivors compared to the non-survivors (SMD at 1.43; 95%CI 0.69-2.17) with the pooled area under the receiver operating characteristic curve (AUC) of 0.76 (95% CI 0.64-0.87) for the mortality prediction. Levels of cfDNA showed a pooled sensitivity of 0.81 (95% CI 0.75-0.86) and specificity of 0.72 (95% CI 0.65-0.78) with pooled diagnostic odd ratio (DOR) at 25.03 (95% CI 5.48-114.43) for the identification of sepsis in critically ill conditions. The cfDNA levels were significantly higher in patients with sepsis and being a helpful indicator for the critically ill conditions of sepsis. Nevertheless, results of the test must be interpreted carefully with the context of all clinical situations.

Neutrophils are the principal trouper of the innate immune system. Activated neutrophils undergo a noble cell death termed NETosis and release a mesh-like structure called neutrophil extracellular traps (NETs) as a part of their defensive strategy against microbial pathogen attack. This web-like architecture includes a DNA backbone embedded with antimicrobial proteins like myeloperoxidase (MPO), neutrophil elastase (NE), histones and deploys in the entrapment and clearance of encountered pathogens. Thus NETs play an inevitable beneficial role in the host's protection. However, recent accumulated evidence shows that dysregulated and enhanced NET formation has various pathological aspects including the promotion of sepsis, pulmonary, cardiovascular, hepatic, nephrological, thrombotic, autoimmune, pregnancy, and cancer diseases, and the list is increasing gradually. In this review, we summarize the NET-mediated pathophysiology of different diseases and focus on some updated potential therapeutic approaches against NETs.

CXCR4hi neutrophils, which are a subset of neutrophils with high CXCR4 expression, are important contributors to sepsis-induced acute lung injury (ALI). PFKFB3, a key glycolysis gene, plays an essential role in neutrophil inflammatory activation. However, the specific involvement of PFKFB3 in sepsis-induced ALI remains unclear. Here, we observed that PFKFB3 was upregulated in CXCR4hi neutrophils and facilitated sepsis-induced ALI. Mechanistically, we observed that PFKFB3 promoted sepsis-induced ALI by enhancing neutrophil extracellular trap (NET) formation by CXCR4hi neutrophils. Further study indicated that PFKFB3 promoted NET formation by upregulating glycolytic metabolism in CXCR4hi neutrophils. In summary, our study uncovered a new mechanism by which CXCR4hi neutrophils trigger sepsis-induced ALI by promoting NET formation, which is supported by PFKFB3-mediated glycolytic metabolism.

Neutrophil extracellular traps (NETs) are large DNA reticular structures secreted by neutrophils and decorated with histones and antimicrobial proteins. As a key mechanism for neutrophils to resist microbial invasion, NETs play an important role in the killing of microorganisms (bacteria, fungi, and viruses). Although NETs are mostly known for mediating microbial killing, increasing evidence suggests that excessive NETs induced by stimulation of physical and chemical components, microorganisms, and pathological factors can exacerbate inflammation and organ damage. This review summarizes the induction and role of NETs in inflammation and focuses on the strategies of inhibiting NETosis and the mechanisms involved in pathogen evasion of NETs. Furthermore, herbal medicine inhibitors and nanodelivery strategies improve the efficiency of inhibition of excessive levels of NETs.

Parental liver transplantation (PLT) improves long-term survival rates in paediatric hepatic failure patients; however, the mechanism of PLT-induced postoperative pulmonary complications (PPCs) is unclear.

A total of 133 paediatric patients undergoing PLT were included. Serum levels of NET components, including circulating free DNA (cfDNA), DNA-histone complex, and myeloperoxidase (MPO)-DNA complex, were detected. The occurrence of PPCs post-PLT, prolonged intensive care unit (ICU) stay and death within one year were recorded as the primary and secondary outcomes.

The overall rate of PPCs in the hospital was 47.4%. High levels of serum cfDNA, DNA-histone complexes and MPO-DNA complexes were associated with an increased risk of PPCs (for cfDNA, OR 2.24; for DNA-histone complex, OR 1.64; and for MPO-DNA, OR 1.94), prolonged ICU stay (OR 1.98, 4.26 and 3.69, respectively), and death within one year (OR 1.53, 2.65 and 1.85, respectively). The area under the curve of NET components for the prediction of PPCs was 0.843 for cfDNA, 0.813 for DNA-histone complexes, and 0.906 for MPO-DNA complexes. During the one-year follow-up, the death rate was higher in patients with PPCs than in patients without PPCs (14.3% vs. 2.9%, P = 0.001).

High serum levels of NET components are associated with an increased incidence of PPCs and death within one year in paediatric patients undergoing PLT. Serum levels of NET components serve as a biomarker for post-PLT PPCs and a prognostic indicator.

The widespread use of orthopedic implants to support or replace bones is increasingly threatened by the risk of incurable bacterial infections, impenetrable microbial biofilms, and irreversible antibiotic resistance. In the past, the development of anti-infective biomaterials focused solely on direct antibacterial properties while ignoring the host's immune response. Inspired by the clearance of infection by the innate neutrophil response and participation in anti-infectious immunity of Zn ions, we report an innovative neutrophil extracellular traps (NETs) strategy, induced by biodegradable pure Zn, which achieved therapeutic efficacy toward biomaterial-related infections. Our in vitro and in vivo data showed that pure Zn was favorable for NETs formation by promoting the release of DNA fibers and granule proteins in a reactive oxygen species (ROS)-dependent manner, thereby retraining and degrading bacteria with an efficiency of up to 99.5%. Transcriptome analysis revealed that cytoskeletal rearrangement and toll-like receptor (TLR) signaling pathway were also involved in Zn-induced NETs formation. Furthermore, the in vivo results of a Staphylococcus aureus (S. aureus)-infected rat model verified that pure Zn potentiated the bactericidal capability of neutrophils around implants, and promoted osseointegration in S. aureus-infected rat femurs. This antibacterial immunity concept lays a foundation for the development of other antibacterial biomaterials and holds great promise for treating orthopedic infections.