Web-like Structures Research Articles

Abstract TP355: Immunohistochemical Confirmation of Neutrophil Extracellular Trap (NET) Enrichment in Stroke Thrombus of Patients with Atrial Fibrillation

Background: Neutrophil Extracellular Traps (NETs) are web-like structures of DNA, histones, and granule proteins that trap pathogens as part of innate immunity but also scaffold red blood cells, fibrin, and platelets in pathological thrombosis. Thrombus NETs have been associated with failed thrombolysis and thrombectomy for acute ischemic stroke (AIS). We previously used mass spectrometry to describe a novel association between NET proteins and atrial fibrillation (AF). This association has not been validated by traditional immunohistochemical methods. Aims/Purpose: We used immunohistochemistry to quantify NETs in AIS thrombus taken from patients with and without comorbid AF. Methods: We selected markers for each of the components of NETS: (1) a granule protein, myeloperoxidase (MPO), (2) citrullinated histone 3 (CitH3), and cellular nucleic acid (NA, using Hoechst 33342). This was a single-blind study using cryopreserved thrombi from 30 patients, 15 with AF. Stroke thrombi were collected over a two-year span, and immediately frozen to -80*C post-thrombectomy. To capture the coverage of NET markers more comprehensively for each patient, we used cryostat to sample four sections (five-micron thickness) from each thrombus. We then fixed sections with 4% formaldehyde in phosphate buffered saline (PBS), performed immunohistological staining, and quantified extent of staining using ImageJ. Data for each NET marker from a single thrombus is represented as the mean percentage of thrombus area staining positively across the four corresponding sections. After performing Shapiro Wilks test for normality, statistical comparisons of NET marker staining coverage between AF and non-AF thrombi were performed with either unpaired T test or Mann-Whitney U test, depending on whether a parametric or non-parametric test was appropriate. Results: Thrombi from patients with AF had greater MPO coverage(17.9 +/- 1.47% versus 11.6 +/- 1.29% in non-AF, p<0.01) and CitH3 coverage(12.6 +/- 1.46% versus 7.4 +/- 1.11% in non-AF, p<0.01). Staining for nucleic acid with Hoechst 33342 trended toward higher coverage in AF but did not reach significance (27.8 +/- 3.35% versus 22.6 +/- 3.20% in non-AF, p=0.30). Conclusion: The present study provides immunohistochemical validation, supporting novel observations in prior proteomic investigations, of increased NETs in stroke thrombi of patients with AF. This can help to identify patient cohorts for future studies of therapies directed at NETs.

Abstract TP383: Neutrophil Extracellular Traps (NETs)-related LncRNAs Regulate Thrombus Formation in Acute Ischemic Stroke Patients with Underlying Medical Conditions

Introduction: Acute cerebral blood flow occlusion by thrombus is a major cause of ischemic stroke. The composition of the resulting thrombus varies depending on the underlying medical condition. Notably, neutrophils, the most abundant white blood cells, play a crucial role in acute ischemic stroke and can form neutrophil extracellular traps (NETs). NETs are web-like structures consisting of DNA, granule proteins, and histones. NETs contribute to thrombo-inflammation and stabilize the thrombus, potentially leading to worse outcomes. While long non-coding RNA (lncRNA) do not encode proteins, they are involved in essential biological processes. The link between lncRNAs and cardiovascular disease is increasingly studied, and their role in ischemic stroke has recently come to attention. We, therefore, hypothesize that lncRNAs within NETs play a critical role in the formation and regulation of thrombi. Method: Human thrombi obtained from endovascular thrombectomy were categorized based on their underlying medical conditions, such as dyslipidemia, hypertension, or a combination of two or more conditions. Frozen sections of the thrombi were immunostained with markers including Citrullinated H3 (CitH3), CD45, neutrophil elastase (NE), and DAPI, and then analyzed using holotomography, confocal microscopy, and spatial transcriptomics. In spatial transcriptomics analysis, NETs and other leukocytes were selected as distal and proximal ROIs for detailed gene expression profiling. The gene expression of HDAC class 1 genes (HDAC1, HDAC2, HDAC3, and HDAC8) was further examined by qPCR. Results: Holotomography analysis revealed that cells in the thrombi of patients with underlying conditions exhibited a high mean refractive index. Immunofluorescence imaging also identified a high percentage of NETs in these samples. Spatial transcriptomics analysis confirmed that NETs in patients with hypertension showed upregulated expression of lncRNAs. Additionally, qPCR results demonstrated increased expression of HDAC class 1 genes, which are associated with NET formation, in patients with underlying conditions. Conclusion: LncRNAs play a crucial role in the formation and regulation of thrombi. Therefore, NETs-related lncRNAs could serve as valuable therapeutic targets and diagnostic markers for acute ischemic stroke patients with underlying diseases.

Transformation of NETs under the effect of pathogens and IgG

Background. Many studies have shown that neutrophil extracellular traps (NETs) in the form of web-like structures are present in the peripheral blood of patients with inflammatory diseases. In our research, in addition to traditional web-like NET structures, several anomalous forms were identified, including NETs with cloud-like appearance.Aim. To investigate morphological and functional transformation of NETs under the influence of Klebsiella pneumoniae and immunoglobulin G (IgG).Materials and methods. The study included 42 patients of Moscow City Clinical Hospital No. 51: 28 patients with acute inflammation in the abdominal cavity (appendicitis, cholecystitis, pancreatitis, peritonitis), 6 patients diagnosed with ulcerative colitis, and 8 patients with hernias. Neutrophils were isolated using gradient-density centrifugation. To calculate NETs, we used SYBR Green I-induced fluorescence microscopy (Evrogen, Russia), with the dye specifically interacting with double-stranded DNA. The functional activity of NETs was determined in the Klebsiella pneumoniae (ATCC 700603) capture test.Results. In patients with inflammatory diseases of the abdominal cavity in the postoperative period, the functional activity of NETs was several times lower than in healthy individuals. NETs in these patients capture and bind no more than 20 cells of the microorganism. Under the effect of IgG, neutrophil networks transform into loose cloud-like structures, which can hardly capture and bind the pathogen, binding only 8.46 ± 0.44 cells of the microorganism. Spontaneous enzymatic degradation of cloud like NETs may be accompanied by the production of secondary alteration factors.Conclusion. The results of the study provide the grounds for the development of new approaches to elaborating vaccination regimens and using immunobiologics that require preliminary monitoring of the state of innate immunity, in particular, neutrophil networks in the patient’s body.

Sepsis-induced NET formation requires MYD88 but is independent of GSDMD and PAD4.

Neutrophils are peripheral blood-circulating leukocytes that play a pivotal role in host defense against bacterial pathogens which upon activation, they release web-like chromatin structures called neutrophil extracellular traps (NETs). Here, we analyzed and compared the importance of myeloid differentiation factor 88 (MYD88), peptidyl arginine deiminase 4 (PAD4), and gasdermin D (GSDMD) for NET formation invivo following sepsis and neutrophilia challenge. Injection of lipopolysaccharide (LPS)/E. coli or the transgenic expression of granulocyte colony-stimulating factor (G-CSF), each induced NET-mediated lethal vascular occlusions in mice with combined genetic deficiency in Dnase1 and Dnase1l3 (D1/D1l3-/-). In accordance with the signaling of toll-like receptors, Myd88/D1/D1l3-/- animals were protected from the formation of lethal intravascular NETs during septic conditions. However, this protection was not observed during neutrophilia. It was unexpected to find that both Gsdmd/D1/D1l3-/- and Pad4/D1/D1l3-/- mice were fully capable of forming NETs upon LPS/E.coli challenge. Sepsis equally triggered a similar inflammatory response in these mice characterized by formation of DNA-rich thrombi, vessel occlusions, and mortality from pulmonary embolism, compared to D1/D1l3-/- mice. Pharmacologic GSDMD inhibitors did not reduce PMA-stimulated NET formation in exvivo models either. Similarly, neither Pad4 nor GSDMD deficiency affected intravascular occlusive NET formation upon neutrophilia challenge. The magnitude of NET production, multi-organ damage, and lethality were comparable to those observed in challenged control mice. In conclusion, our data indicate that NET formation during experimental sepsis and neutrophilia is regulated by distinct stimulus-dependent pathways that may be independent of canonical PAD4 and GSDMD.

Pathophysiological Role of Neutrophil Extracellular Traps in Diet-Induced Obesity and Metabolic Syndrome in Animal Models.

The global pandemic of obesity poses a serious health, social, and economic burden. Patients living with obesity are at an increased risk of developing noncommunicable diseases or to die prematurely. Obesity is a state of chronic low-grade inflammation. Neutrophils are first to be recruited to sites of inflammation, where they contribute to host defense via phagocytosis, degranulation, and extrusion of neutrophil extracellular traps (NETs). NETs are web-like DNA structures of nuclear or mitochondrial DNA associated with cytosolic antimicrobial proteins. The primary function of NETosis is preventing the dissemination of pathogens. However, neutrophils may occasionally misidentify host molecules as danger-associated molecular patterns, triggering NET formation. This can lead to further recruitment of neutrophils, resulting in propagation and a vicious cycle of persistent systemic inflammation. This scenario may occur when neutrophils infiltrate expanded obese adipose tissue. Thus, NETosis is implicated in the pathophysiology of autoimmune and metabolic disorders, including obesity. This review explores the role of NETosis in obesity and two obesity-associated conditions-hypertension and liver steatosis. With the rising prevalence of obesity driving research into its pathophysiology, particularly through diet-induced obesity models in rodents, we discuss insights gained from both human and animal studies. Additionally, we highlight the potential offered by rodent models and the opportunities presented by genetically modified mouse strains for advancing our understanding of obesity-related inflammation.

The occurrences, characteristics, and implications of chaotic rocks (accretionary rocks) within the Trans-Saharan orogenic belt in Nigeria

The present research paper reveals the occurrences of metachaotic rocks/accretionary sediments (mélange) in Igarra, southwest Nigeria. These metachaotic rocks are associated with schistose, marble, pegmatite, granitoid, and gneissose units. The mineral assemblage of the surrounding rocks shows garnet, biotite, staurolite, quartz, and chlorite assemblage, which suggest that the rocks were metamorphosed to the epidote–amphibolite facies grade. The chaotic rocks were classified into four groups based on major differences in color, grain size distribution, and stratigraphic and structural characteristics of observed blocks and matrices surrounding them. Group I metachaotic rocks are melanocratic in color, and form pockets of coarse lineation. They are highly deformed with mullions, rodding structures, and burrow-like cavities and show quartzite veins. Group II occurs as a leucocratic to melanocratic mixture of meta-siltstone and metapelite with greenish rock fragments trapped within the meta-siltstone layers. This unit has a cover of meta-siltstone layers occurring in pockets as striations forming mullion and rodding structures as well as ripple-like lineation. Group III metachaotic rocks occur as multiple benches of different strata tilted between 70 and 80º, with mild folding and burrow-like cavities at the base and well-preserved sedimentary structures. In Group IV, there are evidences of pre-metamorphic soft sediment deformational structures that are well-preserved in the metachaotic rocks, with discontinuous blocks of different units trapped within mixtures of metapelite and meta-siltstone. In this group, micro-faults and quartzite veins are scattered within the rocks. Meta-siltstone occurs as horizontal and vertical layers and web-like structures showing dendritic patterns. Overall, the field and petrographic characteristics suggest the Igarra metachaotic rocks are tectonic in origin and may have been formed within the trench basin of an outer accretionary complex.

Cytokine signalling in formation of neutrophil extracellular traps: Implications for health and diseases.

Neutrophils, as essential component of the innate immune response, form a crucial part in the defence mechanisms through the release of extracellular traps (NETs). These web-like structures, composed of chromatin and antimicrobial proteins, are essential for the entrapment and inactivation of pathogens. However, either constitutive formation or inefficient clearance of NETs leads to adverse effects such as fibrosis, thrombosis, delayed wound healing and tissue damage in multiple diseases associated with sterile inflammation. This dichotomy casts NETs as both protective agents and harmful factors in several diseases such as autoimmune diseases, metabolic syndromes, systemic infections, and malignancies. Besides microbes and their products, variety of stimulants including pro-inflammatory cytokines induce NETs. The complex interactions and cross talk among the pro-inflammatory cytokines including IL-8, IL-6, GM-CSF, TNF-α, IFNs, and IL-1β activate neutrophils to form NETs and also contributes to a vicious circle of inflammatory cascade, leading to increased inflammation, oxidative stress, and thrombotic events. Emerging evidence indicates that the dysregulated cytokine milieus in diseases, such as diabetes mellitus, obesity, atherosclerosis, stroke, rheumatoid arthritis, and systemic lupus erythematosus, potentiate NETs release, thereby promoting disease development. Thus, neutrophils represent both critical effectors and potential therapeutic targets, underscoring their importance in the context of cytokine-mediated therapies for a spectrum of diseases. In the present review, we describe various cytokines and associated signalling pathways activating NETs formation in different human pathologies. Further, the review identifies potential strategies to pharmacologically modulate cytokine pathways to reduce NETs.

Identification of key neutrophil extracellular trap genes in Alzheimer's disease.

Alzheimer's disease (AD) is a neurodegenerative disorder that is associated with neuroinflammation. Neutrophil extracellular traps (NETs) are web-like structures that cause inflammation, but its involvement in AD pathogenesis is unclear. This study aimed to identify key NETs related genes associated with AD. A total of 180 samples from the GSE122063 and GSE36980 dataset were obtained from the GEO repository. The representative genes were obtained, and its diagnostic performance was evaluated by analyzing operating characteristic curves. Consensus clustering and principal component analysis were performed to cluster AD samples. Gene ontology, KEGG pathway enrichment, and protein interaction network were analyzed. Peripheral blood samples were collected from 5 AD patients and 5 healthy donors to determine the expression of representative proteins or genes using WB, ELISA, and RT-qPCR. A total of 297 differentially expressed genes (DEGs) were associated with AD, 18 NETs genes showed potential diagnostic value. NETs genes expression effectively distinguished AD patients into 2 subgroups. The AD1 cluster showed higher abundance of activated dendritic cells, monocytes, and neutrophils, the AD2 cluster showed higher levels of naive B cells, eosinophils, and activated NK cells. We randomly selected and measured the levels representative genes in AD patients. The levels of NETs and CCL2, TLR2 expressions were significantly increased, whereas the level of BDNF was significantly decreased in AD patients comparing with healthy controls. This study identified key NET genes including BDNF, CCL2, and TLR2 to be associated with AD pathogenesis and classification.

Neutrophil extracellular traps behaviors in periodontitis: A mini review

Periodontitis is a multifactorial inflammatory disease brought on by a state of imbalance between the gingival microbiome and the immune responses of the host. Neutrophils are the key cells that play an important role in fighting pathogenic microorganisms in infectious disorders, either by killing them within the cell or secreting antimicrobials attached to large web-like structures known as neutrophil extracellular traps (NETs) through a cell death process known as NETosis. Formation of NETs is important for maintaining homeostasis in periodontal tissues and improvement of health. In periodontitis, an immune-based inflammation impedes NETs formation or causes its destruction, thereby enabling inflammation to progress and destroy periodontal tissues. Polymorphonulear neutrophils (PMNs) inhibit microorganisms and decrease inflammation by secreting of NETs into the pocket environment. Conversely, elevated NET levels can elicit an inflammatory response and provide an environment that is favorable for pathogenic bacteria. NETs biomarkers may prove to be helpful in the diagnosis of periodontitis. However, it is necessary to make future research on the specific mechanism of NETs and the exact interaction between a single bacterium and NETs.

Abstract C014: Degradation of extracellular trap DNA sustains anti-tumor immune responses in breast cancer

Abstract Increased extracellular DNA (exDNA) levels in the blood are a hallmark of metastatic cancer, arising from tumor lysis, apoptosis, necrosis, and neutrophil extracellular traps (NETs) released by tumor-expanded neutrophils. NETs, web-like chromatin structures extruded by neutrophils to trap pathogens, are highly immunogenic due to their DNA and histone content and their persistent interaction with dendritic cells. Tumor cells can also release extracellular traps. In myeloid leukemia, blasts release nuclear content via traps to activate coagulation or sustain myeloproliferation. Extracellular traps have been found in NPM1 mutant AML patients, with mutant NPM co-localizing with histones along exDNA traps. Forcing trap extrusion in NPM mutant leukemia cells has been used to create dendritic cell-based vaccines that break tolerance against NPMc antigens. This suggests that NETs in the tumor microenvironment (TME) may promote tolerance, with breaking tolerance requiring consistent NET formation and dendritic cell interaction. NETs are also seen in solid tumors, such as triple-negative breast cancer (TNBC), where they aid in cancer cell migration, invasion, and awakening of circulating tumor cells. However, the capacity of tumor cells to directly extrude DNA traps is not fully understood. To investigate this, various human and murine breast cancer cell lines were seeded on poly-D-lysine coated slides, stained with DAPI and Sytoxgreen, and observed via confocal microscopy. Tumor cells extruded DNA traps within 4 hours without stimuli. Notably, trap extrusion correlated with cell line aggressiveness and was abolished by DNase, indicating dsDNA composition. The absence of traps in 24-hour cultures suggests DNA extrusion may help rare tumor cells survive and proliferate. In line, DNase treatment reduced tumor proliferation and increased apoptosis. To assess the significance of DNA traps in vivo, BALB/c mice were injected with the highly metastatic 4T1 clone 5 breast cancer model, with or without DNase treatment, and monitored starting 5 days post-injection and weekly until day 28. Histological analysis at the injection site 5 days post-injection showed that DNase treatment reduced both local trap formation and tumor cell proliferation, as indicated by BrdU incorporation. Moreover, immunohistochemistry revealed that in DNase-treated mice, initial tumor growth was followed by complete tumor regression, accompanied by local and systemic CD8 T cell activation. Correspondingly, although a few metastases were detectable at day 14 in DNase-treated mice, no metastases were observed by day 28. Although it remains unclear whether tumor-derived or immune cell-derived traps are most relevant in our model, the results demonstrate that their presence in the TME promotes tolerance despite the inherent immune adjuvant properties of the traps. This suggests that DNase treatment could be particularly effective in combating early tumor development or relapse when used as part of a combination therapy designed to sustain anti-tumor immune responses. Citation Format: Sabina Sangaletti, Paola Portararo, Laura Botti, Valeria Cancila, Claudio Tripodo, Mario P. Colombo, Claudia Chiodoni. Degradation of extracellular trap DNA sustains anti-tumor immune responses in breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C014.

NETosis in autoimmune diseases with focus on psoriasis

Neutrophil granulocytes are a key player in host innate immunity and they provide antimicrobial protection also by NETosis, amongst other mechanisms. NETosis involves the formation of intricate web-like structures, composed of DNA-histone complexes and proteins released by activated neutrophils, commonly referred to as neutrophil extracellular traps (NETs) with sticky and antimicrobial effects on extracellular pathogens. The dysregulation of neutrophils and NETosis may result in autoimmune diseases, due to the formation of autoantibodies, consequent exaggerated immune stimulation, overexpression of various molecules related to NETosis, and infiltration of cutaneous lesions with neutrophils and other immune cell populations. Furthermore, a neutrophil subpopulation was found to be strongly linked to the pathogenesis of these diseases. The interest in the implications of NETosis in autoimmunity has grown in the past years; researchers found new biomarkers and evaluated therapeutics targeted towards this cell death pathway correlated to psoriasis and autoimmunity. Thus, future studies are needed on this matter in order to increase the quality of life in autoimmune disease patients, as NETosis represents a promising therapeutic target. This review article aimed to thoroughly examine neutrophil functions and their association with NETosis in the context of autoimmune diseases, with a particular focus on psoriasis.

How Do ROS Induce NETosis? Oxidative DNA Damage, DNA Repair, and Chromatin Decondensation.

Neutrophil extracellular traps (NETs) are intricate, DNA-based, web-like structures adorned with cytotoxic proteins. They play a crucial role in antimicrobial defense but are also implicated in autoimmune diseases and tissue injury. The process of NET formation, known as NETosis, is a regulated cell death mechanism that involves the release of these structures and is unique to neutrophils. NETosis is heavily dependent on the production of reactive oxygen species (ROS), which can be generated either through NADPH oxidase (NOX) or mitochondrial pathways, leading to NOX-dependent or NOX-independent NETosis, respectively. Recent research has revealed an intricate interplay between ROS production, DNA repair, and NET formation in different contexts. UV radiation can trigger a combined process of NETosis and apoptosis, known as apoNETosis, driven by mitochondrial ROS and DNA repair. Similarly, in calcium ionophore-induced NETosis, both ROS and DNA repair are key components, but only play a partial role. In the case of bacterial infections, the early stages of DNA repair are pivotal. Interestingly, in serum-free conditions, spontaneous NETosis occurs through NOX-derived ROS, with early-stage DNA repair inhibition halting the process, while late-stage inhibition increases it. The intricate balance between DNA repair processes and ROS production appears to be a critical factor in regulating NET formation, with different pathways being activated depending on the nature of the stimulus. These findings not only deepen our understanding of the mechanisms behind NETosis but also suggest potential therapeutic targets for conditions where NETs contribute to disease pathology.

Neutrophil extracellular traps and citrullinated fibrinogen contribute to injury in a porcine model of limb ischemia and reperfusion.

Ischemia/reperfusion injury (IRI) is a complex pathological process, triggered by the restoration of blood flow following an interrupted blood supply. While restoring the blood flow is the only option to salvage the ischemic tissue, reperfusion after a prolonged period of ischemia initiates IRI, triggering a cascade of inflammatory responses ultimately leading to neutrophil recruitment to the inflamed tissue, where they release neutrophil extracellular traps (NETs). NETs are web-like structures of decondensed chromatin and neutrophilic proteins, including peptidyl-arginine deiminase 2 and 4 (PAD2, PAD4), that, once outside, can citrullinate plasma proteins, irreversibly changing their conformation and potentially their function. While the involvement of NETs in IRI is known mainly from rodent models, we aimed to determine the effect of NET formation and especially PADs-mediated extracellular protein citrullination in a porcine model of limb IRI. We conducted our study on amputated pig forelimbs exposed to 1h or 9h of ischemia and then reperfused in vivo for 12h. Limb weight, edema formation, compartmental pressure were measured, and skeletal muscle was analyzed by immunofluorescence (TUNEL assay and dystrophin staining) to evaluate tissue damage. Fibrin tissue deposition, complement deposition and NETs were investigated by immunofluorescence. Citrullinated plasma proteins were immunoprecipitated and citrullinated fibrinogen was identified in the plasma by Western blot and in the tissue by immunofluorescence and Western blot. Our data consolidate the involvement of NETs in a porcine model of limb IRI, correlating their contribution to damage extension with the duration of the ischemic time. We found a massive infiltration of NETs in the group subjected to 9h ischemia compared to the 1h and citrullinated fibrinogen levels, in plasma and tissue, were higher in 9h ischemia group. We propose fibrinogen citrullination as one of the mechanisms contributing to the worsening of IRI. NETs and protein citrullination represent a potential therapeutic target, but approaches are still a matter of debate. Here we introduce the idea of therapeutic approaches against citrullination to specifically inhibit PADs extracellularly, avoiding the downstream effects of hypercitrullination and keeping PADs' and NETs' intracellular regulatory functions.

Breaking barriers: the role of NETosis in blood-brain barrier leakage and age-related cognitive decline

Cognitive impairment is a common age-related comorbidity with blood-brain barrier (BBB) leakage a key event. BBB leakage increases with age, but the mechanisms are still not completely understood. In the current article, we briefly discussed the role of neutrophil extracellular traps (NETs) in age-associated increase in cognitive impairment. NETosis is a process neutrophils release web-like structures called NETs composed of DNA, histones, and antimicrobial proteins. These NETs act as physical barriers to trap and kill pathogens, such as bacteria, viruses, and fungi. Excessive NETs formation has been associated with various pathological conditions such as thrombosis, cancer metastasis, inflammatory diseases, and autoimmune disorders. Recent studies further indicated that NETosis plays a key role in the BBB leakage during stroke and depletion of neutrophils can attenuate the pathology of Alzheimer’s disease (AD) in murine models. In the current article, we briefly discussed the putative role of NETosis in BBB leakage and age-related cognitive impairment. It should briefly summarize the main content of the article, and it may include the background, purpose, significance, methods and conclusions of the article.

Recognition and control of neutrophil extracellular trap formation by MICL

Regulation of neutrophil activation is critical for disease control. Neutrophil extracellular traps (NETs), which are web-like structures composed of DNA and neutrophil-derived proteins, are formed following pro-inflammatory signals; however, if this process is uncontrolled, NETs contribute to disease pathogenesis, exacerbating inflammation and host tissue damage1,2. Here we show that myeloid inhibitory C-type lectin-like (MICL), an inhibitory C-type lectin receptor, directly recognizes DNA in NETs; this interaction is vital to regulate neutrophil activation. Loss or inhibition of MICL functionality leads to uncontrolled NET formation through the ROS–PAD4 pathway and the development of an auto-inflammatory feedback loop. We show that in the context of rheumatoid arthritis, such dysregulation leads to exacerbated pathology in both mouse models and in human patients, where autoantibodies to MICL inhibit key functions of this receptor. Of note, we also detect similarly inhibitory anti-MICL autoantibodies in patients with other diseases linked to aberrant NET formation, including lupus and severe COVID-19. By contrast, dysregulation of NET release is protective during systemic infection with the fungal pathogen Aspergillus fumigatus. Together, we show that the recognition of NETs by MICL represents a fundamental autoregulatory pathway that controls neutrophil activity and NET formation.

Neutrophil extracellular traps in cancer.

Neutrophil extracellular traps (NETs), which consist of chromatin DNA studded with granule proteins, are released by neutrophils in response to bothinfectious and sterile inflammation. Beyond the canonical role in defense against pathogens, the extrusion of NETs also contributes to the initiation, metastasis, and therapeutic response of malignant diseases. Recently, NETs have been implicated in the development and therapeutic responses of various types of tumors. Although extensive work regarding inflammation in tumors has been reported, a comprehensive summary of how these web-like extracellular structures initiate and propagate tumor progression under the specific microenvironment is lacking. In this review, we demonstrate the initiators and related signaling pathways that trigger NETs formation in cancers. Additionally, this review will outline the current molecular mechanisms and regulatory networks of NETs during dormant cancer cells awakening, circulating tumor cells (CTCs) extravasation, and metastatic recurrence of cancer. This is followed by a perspective on the current and potential clinical potential of NETs as therapeutic targets in the treatment of both local and metastatic disease, including the improvement of the efficacy of existing therapies.

The neutrophil extracellular traps in neurological diseases: an update.

Neutrophil extracellular traps (NETs) released by neutrophils are web-like DNA structures adhered to granulin proteins with bactericidal activity and can be an important mechanism for preventing pathogen dissemination or eliminating microorganisms. However, they also play important roles in diseases of other systems, such as the central nervous system. We tracked the latest advances and performed a review based on published original and review articles related to NETs and neurological diseases. Generally, neutrophils barely penetrate the blood-brain barrier into the brain parenchyma, but when pathological changes such as infection, trauma, or neurodegeneration occur, neutrophils rapidly infiltrate the central nervous system to exert their defensive effects. However, neutrophils may adversely affect the host when they uncontrollably release NETs upon persistent neuroinflammation. This review focused on recent advances in understanding the mechanisms and effects of NETs release in neurological diseases, and we also discuss the role of molecules that regulate NETs release in anticipation of clinical applications in neurological diseases.

NETosis-Inspired Cell Surface-Constrained Framework Nucleic Acids Traps (FNATs) for Cascaded Extracellular Recognition and Cellular Behavior Modulation.

Upon pathogenic stimulation, activated neutrophils release nuclear DNA into the extracellular environment, forming web-like DNA structures known as neutrophil extracellular traps (NETs), which capture and kill bacteria, fungi, and cancer cells. This phenomenon is commonly referred to as NETosis. Inspired by this, we introduce a cell surface-constrained web-like framework nucleic acids traps (FNATs) with programmable extracellular recognition capability and cellular behavior modulation. This approach facilitates dynamic key chemical signaling molecule recognition such as adenosine triphosphate (ATP), which is elevated in the extracellular microenvironment, and triggers FNA self-assembly. This, in turn, leads to in situ tightly interwoven FNAs formation on the cell surface, thereby inhibiting target cell migration. Furthermore, it activates a photosensitizer-capturing switch, chlorin e6 (Ce6), and induces cell self-destruction. This cascade platform provides new potential tools for visualizing dynamic extracellular activities and manipulating cellular behaviors using programmable in situ self-assembling DNA molecular devices.

A New Approach for Assessment of Neutrophil Extracellular Traps Through Immunofluorescence Staining in Whole Blood Smears.

Neutrophils, constituting 50%-70% of circulating leukocytes, play crucial roles in host defense and exhibit anti-tumorigenic properties. An elevated peripheral blood neutrophil-to-lymphocyte ratio is associated with decreased survival rates in cancer patients. In response to exposure to various antigens, neutrophils release neutrophil granular proteins, which combine to form web-like structures known as neutrophil extracellular traps (NETs). Previously, the relative percentage of NETs was found to be increased in resected tumor tissue samples from patients with gastrointestinal malignancies. The presence of NETs in peripheral blood is indicative of underlying pathological conditions. Hence, employing a non-invasive method to detect NETs in peripheral blood, along with other diagnostic tests, shows potential as a valuable tool not just for identifying different inflammatory disorders but also for assessing disease severity and determining patient suitability for surgical resection. While reliable methods exist for identifying NETs in tissue, accurately quantifying them in whole blood remains challenging. Many previous methods are time-consuming and rely on a limited set of markers that are inadequate for fully characterizing NETs. Therefore, we established a unique sensitive smear immunofluorescence assay based on blood smears to identify NETs in only as little as 2 μL of whole blood. To identify the NET complexes that have enhanced specificities, this combines the use of various antibodies against neutrophil-specific CD15, NET-specific myeloperoxidase (MPO), citrullinated histone H3 (Cit H3), and nuclear DNA. This protocol offers an easy, affordable, rapid, and non-invasive method for identifying NETs; thus, it can be utilized as a diagnostic marker and targeted through various therapeutic approaches for treating human malignancies. Key features • Characterization of neutrophil extracellular traps in whole blood smears through immunofluorescence staining. • Affordable and quantitative approach to neutrophil extracellular trap detection.

Resveratrol-Ampicillin Dual-Drug Loaded Polyvinylpyrrolidone/Polyvinyl Alcohol Biomimic Electrospun Nanofiber Enriched with Collagen for Efficient Burn Wound Repair.

The healing of burn wounds is a complicated physiological process that involves several stages, including haemostasis, inflammation, proliferation, and remodelling to rebuild the skin and subcutaneous tissue integrity. Recent advancements in nanomaterials, especially nanofibers, have opened a new way for efficient healing of wounds due to burning or other injuries. This study aims to develop and characterize collagen-decorated, bilayered electrospun nanofibrous mats composed of PVP and PVA loaded with Resveratrol (RSV) and Ampicillin (AMP) to accelerate burn wound healing and tissue repair. Nanofibers with smooth surfaces and web-like structures with diameters ranging from 200 to 400 nm were successfully produced by electrospinning. These fibres exhibited excellent in vitro properties, including the ability to absorb wound exudates and undergo biodegradation over a two-week period. Additionally, these nanofibers demonstrated sustained and controlled release of encapsulated Resveratrol (RSV) and Ampicillin (AMP) through in vitro release studies. The zone of inhibition (ZOI) of PVP-PVA-RSV-AMP nanofibers against Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) was found 31±0.09 mm and 12±0.03, respectively, which was significantly higher as compared to positive control. Similarly, the biofilm study confirmed the significant reduction in the formation of biofilms in nanofiber-treated group against both S. aureus and E. coli. X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR) analysis proved the encapsulation of RSV and AMP successfully into nanofibers and their compatibility. Haemolysis assay (%) showed no significant haemolysis (less than 5%) in nanofiber-treated groups, confirmed their cytocompatibility with red blood cells (RBCs). Cell viability assay and cell adhesion on HaCaT cells showed increased cell proliferation, indicating its biocompatibility as well as non-toxic properties. Results of the in-vivo experiments on a burn wound model demonstrated potential burn wound healing in rats confirmed by H&E-stained images and also improved the collagen synthesis in nanofibers-treated groups evidenced by Masson-trichrome staining. The ELISA assay clearly indicated the efficient downregulation of TNF-alpha and IL-6 inflammatory biomarkers after treatment with nanofibers on day 10. The RSV and AMP-loaded nanofiber mats, developed in this study, expedite burn wound healing through their multifaceted approach.