Extracellular Trap Production Research Articles

Characterization of extracellular trap production and release by equine neutrophils in response to different stimuli

This study explores Neutrophil Extracellular Trap (NET) formation in equine neutrophils, which is crucial for eliminating infections and is implicated in various equine inflammatory diseases. We investigated the molecular pathways involved in NET release by equine neutrophils in response to stimuli. We use PMA, A23187, LPS, PAF, OZ, and cytokines, observing NET release in response to PMA, PAF, and A23187. In contrast, LPS, OZ, and the cytokines tested did not induce DNA release or did not consistently induce citrullination of histone 4. Peptidyl-arginine deiminase inhibition completely halted NET release, while NADPH oxidase and mitochondrial reactive oxygen species only played a role in PMA-induced NETs. Neutrophil elastase inhibition modestly affected PAF-induced NET liberation but not in PMA or A23187-induced NET, while myeloperoxidase did not contribute to NET release. We expect to provide a foundation for future investigations into the role of NETs in equine health and disease and the search for potential therapeutic targets.

ß1-adrenergic blockers preserve neuromuscular function by inhibiting the production of extracellular traps during systemic inflammation in mice.

Severe inflammation via innate immune system activation causes organ dysfunction. Among these, the central nervous system (CNS) is particularly affected by encephalopathies. These symptoms are associated with the activation of microglia and a potential infiltration of leukocytes. These immune cells have recently been discovered to have the ability to produce extracellular traps (ETs). While these components capture and destroy pathogens, deleterious effects occur such as reduced neuronal excitability correlated with excessive ETs production. In this study, the objectives were to determine (1) whether immune cells form ETs in the CNS during acute inflammation (2) whether ETs produce neuromuscular disorders and (3) whether an immunomodulatory treatment such as β1-adrenergic blockers limits these effects. We observed an infiltration of neutrophils in the CNS, an activation of microglia and a production of ETs following lipopolysaccharide (LPS) administration. Atenolol, a β1-adrenergic blocker, significantly decreased the production of ETs in both microglia and neutrophils. This treatment also preserved the gastrocnemius motoneuron excitability. Similar results were observed when the production of ETs was prevented by sivelestat, an inhibitor of ET formation. In conclusion, our results demonstrate that LPS administration increases neutrophils infiltration into the CNS, activates immune cells and produces ETs that directly impair neuromuscular function. Prevention of ETs formation by β1-adrenergic blockers partly restores this function and could be a good target in order to reduce adverse effects in severe inflammation such as sepsis but also in other motor related pathologies linked to ETs production.

Composite Hydrogel Modulates Intrinsic Immune-Cascade Neovascularization for Ocular Surface Reconstruction after Corneal Chemical Injury.

Ocular alkali burns recruit neutrophils and triggers neutrophil extracellular trap (NET)-neovascularization cascade effects that limit ocular surface reconstruction and functional repair. However, effective inhibition of the release of neutrophil extracellular traps after a corneal chemical injury, coordination of intrinsic immunity with corneal repair, and exploration of more effective and non-invasive drug-delivery modes are still urgently needed. Using an in vitro coculture system, we found that an alkaline environment stimulates neutrophils to release NETs, which can be regulated by deoxyribonuclease I (DNase I). Inspired by this, we loaded DNase I, which effectively regulates NETs, onto chitosan nanoparticles and combined them with silk fibroin to construct a composite hydrogel that can sustainably regulate NETs. The hydrogel reduced neutrophil extracellular trap production by 50% and neovascularization by approximately 70% through sustained DNase I release after a corneal alkali burn. The complex hydrogel promotes ocular surface reconstruction by modulating the intrinsic immune-cascade neovascularization effect, providing a new research basis for the construction of nanobiomaterials that modulate pathological neovascularization.

A secreted sirtuin from Campylobacter jejuni contributes to neutrophil activation and intestinal inflammation during infection.

Histone modifications control numerous processes in eukaryotes, including inflammation. Some bacterial pathogens alter the activity or expression of host-derived factors, including sirtuins, to modify histones and induce responses that promote infection. In this study, we identified a deacetylase encoded by Campylobacter jejuni which has sirtuin activities and contributes to activation of human neutrophils by the pathogen. This sirtuin is secreted from the bacterium into neutrophils, where it associates with and deacetylates host histones to promote neutrophil activation and extracellular trap production. Using the murine model of campylobacteriosis, we found that a mutant of this bacterial sirtuin efficiently colonized the gastrointestinal tract but was unable to induce cytokine production, gastrointestinal inflammation, and tissue pathology. In conclusion, these results suggest that secreted bacterial sirtuins represent a previously unreported class of bacterial effector and that bacterial-mediated modification of host histones is responsible for the inflammation and pathology that occurs during campylobacteriosis.

Neutrophil extracellular trap production and CCL4L2 expression influence corticosteroid response in asthma.

The association between neutrophil extracellular traps (NETs) and response to inhaled corticosteroids (ICS) in asthma is unclear. To better understand this relationship, we analyzed the blood transcriptomes from children with controlled and uncontrolled asthma in the Taiwanese Consortium of Childhood Asthma Study using weighted gene coexpression network analysis and pathway enrichment methods. We identified 298 uncontrolled asthma-specific differentially expressed genes and one gene module associated with neutrophil-mediated immunity, highlighting a potential role for neutrophils in uncontrolled asthma. We also found that NET abundance was associated with nonresponse to ICS in patients. In a neutrophilic airway inflammation murine model, steroid treatment could not suppress neutrophilic inflammation and airway hyperreactivity. However, NET disruption with deoxyribonuclease I (DNase I) efficiently inhibited airway hyperreactivity and inflammation. Using neutrophil-specific transcriptomic profiles, we found that CCL4L2 was associated with ICS nonresponse in asthma, which was validated in human and murine lung tissue. CCL4L2 expression was also negatively correlated with pulmonary function change after ICS treatment. In summary, steroids fail to suppress neutrophilic airway inflammation, highlighting the potential need to use alternative therapies such as leukotriene receptor antagonists or DNase I that target the neutrophil-associated phenotype. Furthermore, these results highlight CCL4L2 as a potential therapeutic target for individuals with asthma refractory to ICS.

B1-adrenergic blockers preserve motor function by inhibiting the production of extracellular traps

Sepsis is characterized by a dysregulated host response to an infection, potentially leading to fatal organ dysfunctions. Among these organs, the central nervous system (CNS) is particularly affected with encephalopathies associated or not with chronic disabling pain. These symptoms are caused by broad neuroinflammation with activation of resident microglia cells and infiltration of leukocytes, especially neutrophils. These immune cells have the ability to produce extracellular traps (ETs) in blood and tissues. These components are composed of chromatins and various antimicrobial proteins, and its role is to capture and destroy pathogens. However, in case of excessive production, deleterious effects occur such as tissue damage and reduction of neuronal excitability.In this study, the objectives were: 1) to determine whether in a model of endotoxemic shock, neutrophils and microglia form ETs in the CNS; 2) and to determine if a modulation of ETs production was beneficial on neuromuscular disorders associated with endotoxemic shock.We observed an infiltration of neutrophils in the SNC, an activation of microglia and a production of ETs following intraperitoneal lipopolyssacharide (LPS) administration in mice (adult Swiss male, 30-45g). Atenolol, a β1-adrenergic blocker with intrinsic immunomodulatory properties, significantly decreases the production of ETs associated in both microglia and neutrophils (citrullinated histone H3: 279.1 ± 101.9 MFI versus 117.1 ± 77.21 MFI, p<0,01). In addition, this treatment preserved the gastrocnemius motoneurons excitability (39,93 ± 15,16 mV in control group, 8.147 ± 5.892 mV in LPS group and 27.32 ± 10.73 mV in LPS+atenolol group). Similar results have been observed when the production of ETs was prevented by using sivelestat (inhibitor of extracellular traps).In conclusion, our results demonstrate that sepsis increases neutrophil infiltration into the spinal cord, activates immune cells and produces extracellular traps that directly impair motor function. Prevention of ETs formation partly restores motor function and could be a good target in order to reduce adverse effects in sepsis but also in other motor related pathologies. This work was supported by funding from the Chancellerie des Universites de Paris (Legs Poix), INSERM and Paris-Saclay University-UVSQ. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Deciphering the Genetic Code of Autoimmune Kidney Diseases.

Autoimmune kidney diseases occur due to the loss of tolerance to self-antigens, resulting in inflammation and pathological damage to the kidneys. This review focuses on the known genetic associations of the major autoimmune kidney diseases that result in the development of glomerulonephritis: lupus nephritis (LN), anti-neutrophil cytoplasmic associated vasculitis (AAV), anti-glomerular basement disease (also known as Goodpasture's disease), IgA nephropathy (IgAN), and membranous nephritis (MN). Genetic associations with an increased risk of disease are not only associated with polymorphisms in the human leukocyte antigen (HLA) II region, which governs underlying processes in the development of autoimmunity, but are also associated with genes regulating inflammation, such as NFkB, IRF4, and FC γ receptors (FCGR). Critical genome-wide association studies are discussed both to reveal similarities in gene polymorphisms between autoimmune kidney diseases and to explicate differential risks in different ethnicities. Lastly, we review the role of neutrophil extracellular traps, critical inducers of inflammation in LN, AAV, and anti-GBM disease, where inefficient clearance due to polymorphisms in DNase I and genes that regulate neutrophil extracellular trap production are associated with autoimmune kidney diseases.

Protein palmitoylation regulates extracellular vesicle production and function in sepsis.

Extracellular vesicles (EVs) are bioactive membrane-encapsulated particles generated by a series of events involving membrane budding, fission and fusion. Palmitoylation, mediated by DHHC palmitoyl acyltransferases, is a lipidation reaction that increases protein lipophilicity and membrane localization. Here, we report palmitoylation as a novel regulator of EV formation and function during sepsis. Our results showed significantly decreased circulating EVs in mice with DHHC21 functional deficiency (Zdhhc21dep/dep), compared to wild-type (WT) mice 24 h after septic injury. Furthermore, WT and Zdhhc21dep/dep EVs displayed distinct palmitoyl-proteomic profiles. Ingenuity pathway analysis indicated that sepsis altered several inflammation related pathways expressed in EVs, among which the most significantly activated was the complement pathway; however, this sepsis-induced complement enrichment in EVs was greatly blunted in Zdhhc21dep/dep EVs. Functionally, EVs isolated from WT mice with sepsis promoted neutrophil adhesion, transmigration, and neutrophil extracellular trap production; these effects were significantly attenuated by DHHC21 loss-of-function. Furthermore, Zdhhc21dep/dep mice displayed reduced neutrophil infiltration in lungs and improved survival after CLP challenges. These findings indicate that blocking palmitoylation via DHHC21 functional deficiency can reduce sepsis-stimulated production of complement-enriched EVs and attenuates their effects on neutrophil activity.

Lysophosphatidic Acid Receptor 3 Suppress Neutrophil Extracellular Traps Production and Thrombosis During Sepsis.

Sepsis consists of life-threatening organ dysfunction resulting from a dysregulated response to infection. Recent studies have found that excessive neutrophil extracellular traps (NETs) contribute to the pathogenesis of sepsis, thereby increasing morbidity and mortality. Lysophosphatidic acid (LPA) is a small glycerophospholipid molecule that exerts multiple functions by binding to its receptors. Although LPA has been functionally identified to induce NETs, whether and how LPA receptors, especially lysophosphatidic acid receptor 3 (LPA3), play a role in the development of sepsis has never been explored. A comprehensive understanding of the impact of LPA3 on sepsis is essential for the development of medical therapy. After intraperitoneal injection of lipopolysaccharide (LPS), Lpar3 -/-mice showed a substantially higher mortality, more severe injury, and more fibrinogen content in the lungs than wild-type (WT) mice. The values of blood coagulation markers, plasma prothrombin time (PT) and fibrinogen (FIB), indicated that the Lpar3 -/- mice underwent a severe coagulation process, which resulted in increased thrombosis. The levels of NETs in Lpar3 -/- mice were higher than those in WT mice after LPS injection. The mortality rate and degree of lung damage in Lpar3 -/- mice with sepsis were significantly reduced after the destruction of NETs by DNaseI treatment. Furthermore, in vitro experiments with co-cultured monocytes and neutrophils demonstrated that monocytes from Lpar3 -/- mice promoted the formation of NETs, suggesting that LPA3 acting on monocytes inhibits the formation of NETs and plays a protective role in sepsis. Mechanistically, we found that the amount of CD14, an LPS co-receptor, expressed by monocytes in Lpar3 -/-mice was significantly elevated after LPS administration, and the MyD88-p65-NFκB signaling axis, downstream of toll-like receptor 4 signaling, in monocytes was overactivated. Finally, after an injection of the LPA3 agonist (2S)-1-oleoyl-2-methylglycero-3-phosphothionate (OMPT), the survival rate of mice with sepsis was improved, organ damage was reduced, and the production of NETs was decreased. This suggested the possible translational value and application prospects of (2S)-OMPT in the treatment of sepsis. Our study confirms an important protective role of LPA3 in curbing the development of sepsis by suppressing NETs production and thrombosis and provides new ideas for sepsis treatment strategies.

Formation of extracellular traps by circulating neutrophils and monocytes in rheumatoid arthritis patients: a study of new citrullinated autoantigen

Detection of subcellular structures containing typical citrullinated rheumatoid autoantigens in a single compartment presents a special interest, due to importance of anticitrulline autoantibodies for the autoimmune response in RA. Neutrophil and monocyte extracellular traps (NETs and ETs, respectively) may be considered such candidate structures. Our objective was to assess ability of blood neutrophils and monocytes from RA patients to generate NETs and ETs spontaneously and after in vitro induction.32 patients with verified RA and 30 healthy volunteers as controls were included into the study. Circulating neutrophils and monocytes were isolated with one-step density gradient centrifugation using three layers of ficoll-amidotrizoate gradient. Composition of isolated cellular fractions, their viability, and non-specific activation were evaluated microscopically using Trypan Blue exclusion test, as well as Nitro-Blue Tetrazolium test. The NETs were induced by phorbol-12-myristate-13-acetate, and ETs by bacterial LPS. Spontaneous and induced formation of extracellular traps was assessed using fluorescence microscopy. Neutrophil and monocyte fractions contained minute percentages of impurities and low extents of activated and dead cells. Spontaneous NET and ET formation in RA patients was significantly increased comparing to healthy controls. Neutrophils from ACPA-positive RA patients were found to have higher frequency of NET formation, compared to ACPA-negative RA patients. The monocytes did not demonstrate such differences between these subgroups. There were no substantial morphological differences in NETs and ETs patterns between the individuals from both groups. Induced extracellular trap production in RA was significantly higher compared to healthy controls. The level of myeloperoxidase-specific fluorescence in ETs was considerably lower than in NETs. NETs could probably be considered as a source of citrulline autoantigen participating in autoantibody production and stimulation of inflammatory autoimmune responses in RA, whereas ETs may play less important role in this process.

Assessing Respiratory Immune Responses to Haemophilus Influenzae.

Haemophilus influenzae (Hi) is a prevalent bacterium found in a range of respiratory conditions. A variety of different assays/techniques may be used to assess the respiratory immune/inflammatory response to this bacterium. Flow cytometry and confocal microscopy are fluorescence-based technologies that allow detailed characterization of biological responses.Different forms of Hi antigen can be used, including cell wall components, killed/inactivated preparations, and live bacteria. Hi is a fastidious bacterium that requires enriched media but is generally easy to grow in standard laboratory settings. Tissue samples for stimulation with Hi may be obtained from peripheral blood, bronchoscopy, or resected lung (e.g., in patients undergoing surgery for the treatment of lung cancer). Macrophage and neutrophil function may be comprehensively assessed using flow cytometry with a variety of parameters measured, including phagocytosis, reactive oxygen species, and intracellular cytokine production. Lymphocyte function (e.g., T cell and NK cell function) may be specifically assessed using flow cytometry, principally for intracellular cytokine production. Hi infection is a potent inducer of extracellular trap production, both by neutrophils (NETs) and macrophages (METs). Confocal microscopy is arguably the most optimal way to assess NET and MET expression, which may also be used to assess protease activity. Lung immunity to Haemophilus influenzae can be assessed using flow cytometry and confocal microscopy.

Molecular and Cellular Biomarkers of COVID-19 Prognosis: Protocol for the Prospective Cohort TARGET Study.

BackgroundSince the beginning of the COVID-19 pandemic, the world’s attention has been focused on better understanding the relation between the human host and the SARS-CoV-2 virus, as its action has led to hundreds of thousands of deaths.ObjectiveIn this context, we decided to study certain consequences of the abundant cytokine release over the innate and adaptive immune systems, inflammation, and hemostasis, comparing mild and severe forms of COVID-19.MethodsTo accomplish these aims, we will analyze demographic characteristics, biochemical tests, immune biomarkers, leukocyte phenotyping, immunoglobulin profile, hormonal release (cortisol and prolactin), gene expression, thromboelastometry, neutralizing antibodies, metabolic profile, and neutrophil function (reactive oxygen species production, neutrophil extracellular trap production, phagocytosis, migration, gene expression, and proteomics). A total of 200 reverse transcription polymerase chain reaction–confirmed patients will be enrolled and divided into two groups: mild/moderate or severe/critical forms of COVID-19. Blood samples will be collected at different times: at inclusion and after 9 and 18 days, with an additional 3-day sample for severe patients. We believe that this information will provide more knowledge for future studies that will provide more robust and useful clinical information that may allow for better decisions at the front lines of health care.ResultsThe recruitment began in June 2020 and is still in progress. It is expected to continue until February 2021. Data analysis is scheduled to start after all data have been collected. The coagulation study branch is complete and is already in the analysis phase.ConclusionsThis study is original in terms of the different parameters analyzed in the same sample of patients with COVID-19. The project, which is currently in the data collection phase, was approved by the Brazilian Committee of Ethics in Human Research (CAAE 30846920.7.0000.0008).Trial RegistrationBrazilian Registry of Clinical Trials RBR-62zdkk; https://ensaiosclinicos.gov.br/rg/RBR-62zdkkInternational Registered Report Identifier (IRRID)DERR1-10.2196/24211

Neutrophil extracellular traps-periodontal implications of netosis: A literature review

Neutrophils are the primary line of innate immune defense against infectious diseases. Numerous researches in the field of neutrophil cell biology explain their role in skewing the immune response in various infections or inflammatory disorders revealed their importance within the regulation of immune response. It has been demonstrated that neutrophils release neutrophil extracellular traps (NETs) for the containment of infection and inflammation. NETosis, being an alternate style of death is that the creation of web-like chromatin decondensates by suitably primed neutrophils as a response to stimulus geared toward containing and eliminating the identical. NETs are implicated in periodontitis and COVID-19 as both these disease form a state of inflammation and be a source of constant harm to the tissues involved. This review thereby highlights the emerging role of neutrophil extracellular trap production in the regulation of immune response and its implication in periodontal disease predisposing towards adverse COVID-19 related outcomes.

Hemocyte extracellular traps of Manila clam Ruditapes philippinarum: Production characteristics and antibacterial effects

Extracellular traps (ETs) have been found to be an important strategy of mammals to immobilize and kill invading microorganisms. In the present study, we observed the formation of ETs in the hemocytes of marine mollusks Ruditapes philippinarum in response to challenge from bacteria Vibrio anguillarum, and examined the potential factors and signaling pathways underling this process. We detected an increase of reactive oxygen species (ROS) and myeloperoxidase (MPO) production during ETosis, accompanied by significantly up-regulated expression of ROS-related and MPO genes. The suppression of ETs structures by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor (diphenyleneiodonium chloride, DPI) and MPO inhibitor (aminobenzoic acid hydrazide, ABAH) further confirmed the essential roles ROS and MPO played in ETosis. Furthermore, ET production could be inhibited by phosphotidylinsitol-3-kinase (PI3K) inhibitor (LY294002) and extracellular regulated protein kinase (ERK) inhibitor (U0126), suggesting the idea that both the PI3K and ERK pathways were suggested to function during ETosis. In addition, the ETosis process was accompanied by enhancement of glycolysis-related enzymatic activities, e.g., pyruvate kinase (PK) and hexokinase (HK), and over-expression of the glycolysis-related genes, e.g., PK, HK and glucose transport protein (GLUT), indicating high involvement of glycolysis in the ETosis process. Furthermore, our scanning electron microscopy (SEM) observation and antibacterial activities test successfully showed the patterns how clam ETs entrapped and killed the invading V. anguillarum. Taken together, our results revealed that ETosis with bactericidal effect increased ROS, MPO and glycolysis level and carried out in a ROS-, MPO-, PI3K-ERK-dependent manner.

Extracellular traps (ETosis) can be activated through NADPH-dependent and -independent mechanisms in bivalve mollusks

In mammals, NETosis is a process in which the activation of neutrophils induces the release of chromatin. This DNA prevents the spread of infection by trapping, neutralizing and killing microorganisms during their interaction with antimicrobial proteins. The release of NETs is usually triggered by stimuli that promote reactive oxygen species production. Although this release of extracellular traps (ETs) has been described in some groups of invertebrates, there is a lack of basic information about them in these animals. In the present study, we describe a robust and reproducible model for the induction, analysis and quantification of ETs production using hemocytes from the bivalve Mytilus galloprovincialis. We analyzed the structure of ETs and the involvement of the ROS in the activation of this process. It was demonstrated that the formation of ETs in hemocytes can be triggered through NOX-dependent and NOX-independent pathways, depending on the stimuli used.

Neutrophil GPCRs as novel therapeutic targets

Each year in the United States, 2 million people are infected with antibiotic resistant pathogens, such as methicillin‐resistant Staphylococcus aureus (MRSA); 23,000 patients die as a result of such infections. With the growth of antibiotic resistance and the slowing in the development of new antibiotics, the discovery of new, effective approaches to treat infections is an important unmet medical need. G protein‐coupled receptors (GPCRs) in polymorphonuclear neutrophils (PMNs), the first‐line effectors of the innate immune response, are a novel but unexplored potential class of “immune boosting” targets. Using a combination of qPCR based arrays and RNAseq analysis, we defined and quantified GPCR expression in human PMNs and found 174 GPCRs commonly expressed among healthy donors tested. To help determine which GPCRs might be involved in response to pathogens, we assessed the GPCR profile of PMNs incubated with MRSA for 3 h. We found numerous GPCRs whose expression increased at least 2‐fold upon pathogen exposure. Analyses of PMN function (e.g., chemotaxis, extracellular trap production, bacterial killing) using selective agonists/antagonists of several such GPCRs indicated that pharmacological agents can “boost” the function of PMNs, including the killing of bacteria. The results indicate that: 1) Human PMNs express a large profile of GPCRs, expression of a number of which is altered by exposure to pathogenic bacteria. 2) Previously unrecognized PMN‐expressed GPCRs can boost PMN function, including the ability to kill bacteria. These data thus suggest a new paradigm for enhancement of innate immunity: targeting of neutrophil‐expressed GPCRs as a means to treat bacterial infections, including ones caused by antibiotic‐resistant bacteria. Furthermore, such GPCRs may also represent new therapeutic targets for the treatment of inflammatory disorders driven by aberrant neutrophil function.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Augmentation of Urinary Lactoferrin Enhances Host Innate Immune Clearance of Uropathogenic Escherichia coli

Urinary tract infection (UTI) is a prominent global health care burden. Although UTI is readily treated with antibiotics in healthy adults, complicated cases in immune-compromised individuals and the emerging antibiotic resistance of several uropathogens have accelerated the need for new treatment strategies. Here, we surveyed the composition of urinary exosomes in a mouse model of uropathgenic Escherichia coli (UPEC) UTI to identify specific urinary tract defense constituents for therapeutic development. We found an enrichment of the iron-binding glycoprotein lactoferrin in the urinary exosomes of infected mice. In subsequent in vitro studies, we identified human bladder epithelial cells as a source of lactoferrin during UPEC infection. We further established that exogenous treatment with human lactoferrin (hLf) reduces UPEC epithelial adherence and enhances neutrophil antimicrobial functions including bacterial killing and extracellular trap production. Notably, a single intravesicular dose of hLf drastically reduced bladder bacterial burden and neutrophil infiltration in our murine UTI model. We propose that lactoferrin is an important modulator of innate immune responses in the urinary tract and has potential application in novel therapeutic design for UTI.

Neutrophil extracellular trap production contributes to pathogenesis in SIV-infected nonhuman primates.

Neutrophil extracellular traps (NETs) are involved in the pathogenesis of many infectious diseases, yet their dynamics and impact on HIV/SIV infection have not yet been assessed. We hypothesized that SIV infection and the related microbial translocation trigger NET activation and release (NETosis), and we investigated the interactions between NETs and immune cell populations and platelets. We compared and contrasted the levels of NETs between SIV-uninfected, SIV-infected, and SIV-infected antiretroviral-treated nonhuman primates. We also cocultured neutrophils from these animals with either peripheral blood mononuclear cells or platelets. Increased NET production was observed throughout SIV infection. In chronically infected animals, NETs were found in the gut, lung, and liver, and in the blood vessels of kidney and heart. Antiretroviral therapy (ART) decreased NETosis, albeit above preinfection levels. NETs captured CD4+ and CD8+ T cells, B cells, and monocytes, irrespective of their infection status, potentially contributing to the indiscriminate generalized immune cell loss characteristic to HIV/SIV infection, and limiting the CD4+ T cell recovery under ART. By capturing and facilitating aggregation of platelets, and through expression of increased tissue factor levels, NETs may also enhance HIV/SIV-related coagulopathy and promote cardiovascular comorbidities.

Control of Mucosal Candidiasis in the Zebrafish Swim Bladder Depends on Neutrophils That Block Filament Invasion and Drive Extracellular-Trap Production.

Candida albicans is a ubiquitous mucosal commensal that is normally prevented from causing acute or chronic invasive disease. Neutrophils contribute to protection in oral infection but exacerbate vulvovaginal candidiasis. To dissect the role of neutrophils during mucosal candidiasis, we took advantage of a new, transparent zebrafish swim bladder infection model. Intravital microscopic tracking of individual animals revealed that the blocking of neutrophil recruitment leads to rapid mortality in this model through faster disease progression. Conversely, artificial recruitment of neutrophils during early infection reduces disease pressure. Noninvasive longitudinal tracking showed that mortality is a consequence of C. albicans breaching the epithelial barrier and invading surrounding tissues. Accordingly, we found that a hyperfilamentous C. albicans strain breaches the epithelial barrier more frequently and causes mortality in immunocompetent zebrafish. A lack of neutrophils at the infection site is associated with less fungus-associated extracellular DNA and less damage to fungal filaments, suggesting that neutrophil extracellular traps help to protect the epithelial barrier from C. albicans breach. We propose a homeostatic model where C. albicans disease pressure is balanced by neutrophil-mediated damage of fungi, maintaining this organism as a commensal while minimizing the risk of damage to host tissue. The unequaled ability to dissect infection dynamics at a high spatiotemporal resolution makes this zebrafish model a unique tool for understanding mucosal host-pathogen interactions.

Neutrophil Extracellular Trap Production in Patients with Colorectal Cancer In Vitro

Purpose Neutrophil Extracellular Traps (NETs) are extracellular neutrophil derived DNA webs which have been implicated in cancer progression and in the development of metastases. NETs production in patients with colorectal cancer was investigated to elucidate their role and prognostic significance. Methods Systemic neutrophils were isolated from consecutive patients with colorectal cancer and from age-matched healthy volunteers. Neutrophils were stimulated to produce NETs which were quantified by a measure of the fluorescence of the extracellular DNA. The impact of cancer location, tumour stage, and patient outcomes (complications, length of stay, and mortality) on NET production was investigated. Results Quantification of NET formation was performed in patients with colorectal cancer (n = 45) and in well-matched healthy individuals (n = 20). Significant increases in NETs production in response to no stimulant (9,735 AFU versus 11347 AFU, p = 0.0209), IL-8 (8,644 AFU versus 11,915 AFU, p = 0.0032), and LPS (10,576 AFU versus 12,473 AFU, p = 0.0428) were identified in patients with colorectal cancer. A significant increase in NETs production in response to fMLP was detected in patients who developed significant postoperative complications (11,760 AFU versus 18,340 AFU, p = 0.0242) and who had a prolonged hospital recovery (9,008 AFU versus 12,530 AFU, p = 0.0476). An increase in NETs production was also observed in patients who died, but this did not reach statistical significance. Cancer location and tumour stage did not appear to affect preoperative NETs production. Conclusions Patients with colorectal cancer have significantly increased NETs production in vitro when compared to healthy volunteers, possibly implicating them in cancer development. Adverse patient outcomes were associated with increased preoperative NETs production, which highlights them as potential therapeutic targets.