Neutrophil Elastase Release Research Articles

Malondialdehyde-specific natural IgM inhibit toll-like receptor 4 and peptidyl-arginine deiminase 4-dependent NETosis triggered by coronary extracellular vesicles of myocardial infarction patients

Abstract Background Neutrophil extracellular traps (NETs) are critical mediators of thromboinflammation during acute myocardial infarction (AMI). However, triggers and signalling pathways of NETosis in AMI remain incompletely understood. Levels of extracellular vesicles (EV) carrying oxidation-specific epitopes (OSE) originating from lipid peroxidation are increased at the culprit site in AMI. Importantly, natural IgM antibodies with specificity for OSE, such as malondialdehyde (MDA), have been shown to modulate functional effects of EV, and are associated with reduced cardiovascular risk. Purpose We investigated the stimulatory capacity of EV on neutrophil effector functions and specifically targeted key mediators of NET formation using pharmacological inhibitors and atheroprotective natural IgM to inhibit this process. Methods Patients were included after diagnosis of ST-segment elevation myocardial infarction and blood was aspirated from the culprit site and peripheral arterial site (n=28) during primary percutaneous coronary intervention (pPCI). Myocardial function was documented by cardiac magnetic resonance imaging 4±2 days and 195±15 days after pPCI. EV were isolated from cell culture supernatants and culprit site plasma for neutrophil stimulation in vitro. Pharmacological inhibitors were used to map NET signalling pathways of EV. Isolated EV were used for neutrophil stimulation in a murine injection model in the presence of the MDA-specific IgM LR04 or an isotype control. NET formation and other neutrophil functions were assessed by flow cytometry, ELISA and fluorescence microscopy. Results Levels of NET surrogate markers and CD45+ MDA+ EV were higher at the culprit site than in the peripheral circulation. EV generated by LPS-activated THP-1 monocytic cells induced in vitro NET formation, release of neutrophil elastase and IL-8, and degranulation of MPO and NGAL in primary human neutrophils, but not reactive oxygen species. Toll-like receptor 4 (TLR4) and peptidyl-arginine deiminase 4 (PAD4) were identified as key mediators of NET formation induced by in vitro-generated EV and EV isolated from the culprit site of AMI patients. Functionally, the MDA-specific monoclonal IgM antibody LR04, but not an isotype control, inhibited the ability of patient-derived and in vitro-generated EV to trigger release of NETs in primary human neutrophils and in mice. Titres of MDA-specific IgM antibodies were inversely associated with the NET marker citH3 in blood of AMI patients. Finally, we found that the concentration of CD45+ MDA+ EV per protective MDA-specific IgM at the culprit site showed an inverse association with left ventricular ejection fraction 72 hours and 6 months after AMI. Conclusions We show that EV can trigger several neutrophil effector functions. EV-induced NET formation is TLR4- and PAD4-dependent and can be inhibited by OSE-specific natural IgM potentially influencing cardiovascular outcomes after an acute event.

Blocking HXA3-mediated neutrophil elastase release during S. pneumoniae lung infection limits pulmonary epithelial barrier disruption and bacteremia.

Streptococcus pneumoniae (Sp), a leading cause of community-acquired pneumonia, can spread from the lung into the bloodstream to cause septicemia and meningitis, with a concomitant threefold increase in mortality. Limitations in vaccine efficacy and a rise in antimicrobial resistance have spurred searches for host-directed therapies that target pathogenic immune processes. Polymorphonuclear leukocytes (PMNs) are essential for infection control but can also promote tissue damage and pathogen spread. The major Sp virulence factor, pneumolysin, triggers acute inflammation by stimulating the 12-lipoxygenase (12-LOX) eicosanoid synthesis pathway in epithelial cells. This pathway is required for systemic spread in a mouse pneumonia model and produces a number of bioactive lipids, including hepoxilin A3 (HXA3), a hydroxy epoxide PMN chemoattractant that has been hypothesized to facilitate breach of mucosal barriers. To understand how 12-LOX-dependent inflammation promotes dissemination during Sp lung infection and dissemination, we utilized bronchial stem cell-derived air-liquid interface cultures that lack this enzyme to show that HXA3 methyl ester (HXA3-ME) is sufficient to promote basolateral-to-apical PMN transmigration, monolayer disruption, and concomitant Sp barrier breach. In contrast, PMN transmigration in response to the non-eicosanoid chemoattractant N-formyl-L-methionyl-L-leucyl-phenylalanine (fMLP) did not lead to epithelial disruption or bacterial translocation. Correspondingly, HXA3-ME but not fMLP increased the release of neutrophil elastase (NE) from Sp-infected PMNs. Pharmacologic blockade of NE secretion or activity diminished epithelial barrier disruption and bacteremia after pulmonary challenge of mice. Thus, HXA3 promotes barrier-disrupting PMN transmigration and NE release, pathological events that can be targeted to curtail systemic disease following pneumococcal pneumonia.IMPORTANCEStreptococcus pneumoniae (Sp), a leading cause of pneumonia, can spread from the lung into the bloodstream to cause systemic disease. Limitations in vaccine efficacy and a rise in antimicrobial resistance have spurred searches for host-directed therapies that limit pathologic host immune responses to Sp. Excessive polymorphonuclear leukocyte (PMN) infiltration into Sp-infected airways promotes systemic disease. Using stem cell-derived respiratory cultures that reflect bona fide lung epithelium, we identified eicosanoid hepoxilin A3 as a critical pulmonary PMN chemoattractant that is sufficient to drive PMN-mediated epithelial damage by inducing the release of neutrophil elastase. Inhibition of the release or activity of this protease in mice limited epithelial barrier disruption and bacterial dissemination, suggesting a new host-directed treatment for Sp lung infection.

Blocking HXA3-mediated neutrophil elastase release during S. pneumoniae lung infection limits pulmonary epithelial barrier disruption and bacteremia.

Streptococcus pneumoniae (Sp), a leading cause of community-acquired pneumonia, can spread from the lung into the bloodstream to cause septicemia and meningitis, with a concomitant three-fold increase in mortality. Limitations in vaccine efficacy and a rise in antimicrobial resistance have spurred searches for host-directed therapies that target pathogenic immune processes. Polymorphonuclear leukocytes (PMNs) are essential for infection control but can also promote tissue damage and pathogen spread. The major Sp virulence factor, pneumolysin (PLY), triggers acute inflammation by stimulating the 12-lipoxygenase (12-LOX) eicosanoid synthesis pathway in epithelial cells. This pathway is required for systemic spread in a mouse pneumonia model and produces a number of bioactive lipids, including hepoxilin A3 (HXA3), a hydroxy epoxide PMN chemoattractant that has been hypothesized to facilitate breach of mucosal barriers. To understand how 12-LOX-dependent inflammation promotes dissemination during Sp lung infection and dissemination, we utilized bronchial stem cell-derived air-liquid interface (ALI) cultures that lack this enzyme to show that HXA3 methyl ester (HXA3-ME) is sufficient to promote basolateral-to-apical PMN transmigration, monolayer disruption, and concomitant Sp barrier breach. In contrast, PMN transmigration in response to the non-eicosanoid chemoattractant fMLP did not lead to epithelial disruption or bacterial translocation. Correspondingly, HXA3-ME but not fMLP increased release of neutrophil elastase (NE) from Sp-infected PMNs. Pharmacologic blockade of NE secretion or activity diminished epithelial barrier disruption and bacteremia after pulmonary challenge of mice. Thus, HXA3 promotes barrier disrupting PMN transmigration and NE release, pathological events that can be targeted to curtail systemic disease following pneumococcal pneumonia.

Inhibition of neutrophil elastase by the bacterial serine protease inhibitor ecotin in cystic fibrosis airway samples

Abstract Cystic fibrosis (CF), a prevalent fatal genetic condition in North America, disrupts respiratory function by impeding chloride ion transport across mucosal surfaces. This leads to the thickening of mucus, resulting in constant lung inflammation and elevated levels of neutrophils. Neutrophil elastase (NE), a potent serine protease crucial for host defense, is released by these neutrophils. However, in excess, NE can cause detrimental host tissue damage. Abundant in CF airways, NE is a key predictor of lung disease progression. While past studies have explored targeting NE in CF, no optimal inhibitors have reached clinics. Our study tested ecotin, a bacterial periplasmic serine protease inhibitor, for its potential to impede NE activity in CF airway samples and curb NE release from human neutrophils. Results show that ecotin significantly reduces NE activity in sputum samples obtained from several CF patients. Additionally, we found that ecotin inhibits NE release from neutrophils exposed to CF lung stimuli like Staphylococcus aureus bacteria. These results establish ecotin as a promising NE inhibitor in CF with clinical potential.

The pathological function of neutrophils in pemphigoid diseases

Abstract Pemphigoid diseases (PDs) are a group of autoimmune blistering diseases, including bullous pemphigoid, epidermolysis bullosa acquisita, mucous membrane pemphigoid, linear immunoglobulin A disease, and other rare variants. These diseases are characterized by the presence of autoantibodies that target proteins at the dermal-epidermal junction, resulting in the formation of tense blisters and erosions on the skin and/or mucosa. The current therapeutic approaches, such as systemic corticosteroid, are associated with significant adverse effects, highlighting that safer and more effective treatment options are an urgent clinical need. To address this unmet need, a comprehensive understanding of the detailed mechanisms underlying PDs is essential. Based on their histopathological infiltration in pemphigoid lesions, neutrophils have long been implicated as major contributors to the initiation and progression of the diseases. Numerous in vivo and in vitro studies have investigated the role of neutrophils in the pemphigoid pathology, revealing various pathological mechanisms induced by these cells, including the release of neutrophil elastase and matrix metalloproteinase-9, as well as the formation of neutrophil extracellular traps. The present review provides a comprehensive summary and critical evaluation of the current understanding regarding the role of neutrophils in PDs. In addition, it discusses the potential of targeting neutrophil-associated pathways as a novel therapeutic approach for the diseases.

Granular Insights: Neutrophil Predominance and Elastase Release in Severe Asthma Exacerbations in a Pediatric Cohort.

The chronic inflammatory component of asthma is propagated by granulocytes, including neutrophils and eosinophils, in the peripheral circulation and airway. Previous studies have suggested that these cells have an altered expression of adhesion-related molecules and a propensity for the release of granule contents that may contribute to tissue damage and enhance inflammatory complications in patients with status asthmaticus. The goal of this prospective cohort study at a tertiary care pediatric hospital with a large population of asthma patients was to assess the role of granulocyte-based inflammation in the development of asthma exacerbation. Subjects were enrolled from two patient populations: those with mild-to-moderate asthma exacerbations seen in the emergency department and those with severe asthma admitted to the intensive care unit (PICU). Clinical data were collected, and blood was drawn. Granulocytes were immediately purified, and the phenotype was assessed, including the expression of cell surface markers, elastase release, and cytokine production. Severe asthmatics admitted to the PICU displayed a significantly higher total neutrophil count when compared with healthy donors. Moreover, little to no eosinophils were found in granulocyte preparations from severe asthmatics. Circulating neutrophils from severe asthmatics admitted to the PICU displayed significantly increased elastase release ex vivo when compared with the PMN from healthy donors. These data suggest that the neutrophil-based activation and release of inflammatory products displayed by severe asthmatics may contribute to the propagation of asthma exacerbations.

A Serological Neoepitope Biomarker of Neutrophil Elastase-Degraded Calprotectin, Associated with Neutrophil Activity, Identifies Idiopathic Pulmonary Fibrosis and Chronic Obstructive Pulmonary Disease More Effectively Than Total Calprotectin.

Neutrophil activation can release neutrophil extracellular traps (NETs) in acute inflammation. NETs result in the release of human neutrophil elastase (HNE) and calprotectin, where the former can degrade the latter and generate protein fragments associated with neutrophil activity. We investigated this in chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF) using the novel neoepitope biomarker CPa9-HNE, quantifying a specific HNE-mediated fragment of calprotectin in serum. CPa9-HNE was compared to total calprotectin. Initially, CPa9-HNE was measured in healthy (n = 39), COPD (n = 67), and IPF (n = 16) serum using a neoepitope-specific competitive enzyme-linked immunosorbent assay. Then, a head-to-head comparison of CPa9-HNE and total calprotectin, a non-neoepitope, was conducted in healthy (n = 19), COPD (n = 25), and IPF (n = 19) participants. CPa9-HNE levels were significantly increased in COPD (p < 0.0001) and IPF subjects (p = 0.0001) when compared to healthy participants. Additionally, CPa9-HNE distinguished IPF (p < 0.0001) and COPD (p < 0.0001) from healthy participants more effectively than total calprotectin for IPF (p = 0.0051) and COPD (p = 0.0069). Here, CPa9-HNE also distinguished IPF from COPD (p = 0.045) participants, which was not observed for total calprotectin (p = 0.98). Neutrophil activity was significantly higher, as assessed via serum CPa9-HNE, for COPD and IPF compared to healthy participants. Additionally, CPa9-HNE exceeded the ability of non-neoepitope calprotectin serum measurements to separate healthy from lung disease and even COPD from IPF participants, indicating that neutrophil activity is essential for both COPD and IPF.

Pharmacological inhibition of the cysteine protease cathepsin C improves graft function after heart transplantation in rats

BackgroundHeart transplantation (HTX) is the standard treatment for end-stage heart failure. However, reperfusion following an ischemic period can contribute to myocardial injury. Neutrophil infiltration, along with the subsequent release of tissue-degrading neutrophil elastase (NE)-related serine proteases and oxygen-derived radicals, is associated with adverse graft outcomes. The inhibition of cathepsin C (CatC) has been shown to block NE-related protease activation. We hypothesized that the CatC inhibitor BI-9740 improves graft function after HTX.MethodsIn a rat model of HTX, the recipient Lewis rats were orally administered with either a placebo (n = 12) or BI-9740 (n = 11, 20 mg/kg) once daily for 12 days. Donor hearts from untreated Lewis rats were explanted, preserved in a cardioplegic solution, and subsequently heterotopically implanted. In vivo left-ventricular (LV) graft function was assessed after 1 h of reperfusion. The proteolytic activity of neutrophil serine proteases was determined in bone marrow lysates from BI-9740-treated and control rats. Additionally, myocardial morphological changes were examined, and heart samples underwent immunohistochemistry and western blot analysis.ResultsThe NE-related proteolytic activity in bone marrow cell lysates was markedly decreased in the BI-9740-treated rats compared to those of the placebo group. Histopathological lesions, elevated CatC and myeloperoxidase-positive cell infiltration, and nitrotyrosine immunoreactivity with an increased number of poly(ADP-ribose) polymerase (PARP)-1-positive cells were lowered in the hearts of animals treated with BI-9740 compared to placebo groups. Regarding the functional parameters of the implanted graft, improvements were observed in both systolic function (LV systolic pressure 110 ± 6 vs 74 ± 6 mmHg; dP/dtmax 2782 ± 149 vs 2076 ± 167 mmHg/s, LV developed pressure, at an intraventricular volume of 200 µl, p < 0.05) and diastolic function in the hearts of BI-9740 treated animals compared with those receiving the only placebo. Furthermore, the administration of BI-9740 resulted in a shorter graft re-beating time compared to the placebo group. However, this study did not provide evidence of DNA fragmentation, the generation of both superoxide anions and hydrogen peroxide, correlating with the absence of protein alterations related to apoptosis, as evidenced by western blot in grafts after HTX.ConclusionsWe provided experimental evidence that pharmacological inhibition of CatC improves graft function following HTX in rats.

Aberrant neutrophil degranulation in hospitalized patients with COVID-19 partially remains for 6 months.

Neutrophils are important players in COVID-19, contributing to tissue damage by release of inflammatory mediators, including ROS and neutrophil elastase. Longitudinal studies on the effects of COVID-19 on neutrophil phenotype and function are scarce. Here, we longitudinally investigated the phenotype and degranulation of neutrophils in COVID-19 patients (28 nonhospitalized and 35 hospitalized patients) compared with 17 healthy donors (HDs). We assessed phenotype, degranulation, CXCL8 (IL-8) release, and ROS generation within 8 days, at one or 6 month(s) after COVID-19 diagnosis. For degranulation and ROS production, we stimulated neutrophils, either with ssRNA and TNF or granulocyte-macrophage colony-stimulating factor and N-Formylmethionyl-leucyl-phenylalanine. During active COVID-19, neutrophils from hospitalized patients were more immature than from HDs and were impaired in degranulation and ROS generation, while neutrophils from nonhospitalized patients only demonstrated reduced CD66b+ granule release and ROS production. Baseline CD63 expression, indicative of primary granule release, and CXCL8 production by neutrophils from hospitalized patients were elevated for up to 6 months. These findings show that patients hospitalized due to COVID-19, but not nonhospitalized patients, demonstrated an aberrant neutrophil phenotype, degranulation, CXCL8 release, and ROS generation that partially persists up to 6 months after infection.

Effect of Acute High-Intensity Interval Training on Immune Function and Oxidative Stress in Canoe/Kayak Athletes.

The aim of this study was to investigate the effects of acute high-intensity interval training (HIIT) on immune function and oxidative stress in male canoe/kayak athletes who were well trained. A total of 22 participants were voluntarily recruited with an age range of 15.9 ± 2.3 years, height of 172.2 ± 5.5 cm, body mass of 63.30 ± 6.95 kg, and body fat of 13.77 ± 3.76%. The modified Wingate kayaking test on a kayak ergometer was performed by all participants. Blood samples were collected at three different time points: before the test (Pre-T), immediately after (Post-T), and 3 h post-test (Post-3 h). Saliva samples were collected at two different time points: before the test (Pre-T) and 3 h after the test (Post-3 h). Results indicated that acute canoe/kayak ergometry HIIT had significant effects on the percentages and counts of leukocytes, neutrophils, lymphocytes, and lymphocyte subsets. Additionally, it resulted in increased total LPS-stimulated neutrophil elastase release and alterations in plasma concentrations of superoxide dismutase, catalase, and TBARS. These findings suggest that conventional kayak HIIT regimens can have short-term effects on immune function and induce oxidative stress in athletes.

Reduction of neutrophil extracellular traps accelerates inflammatory resolution and increases bone formation on titanium implants.

Neutrophils are the most abundant immune cells in the blood and the first cells to be recruited to the biomaterial implantation site. Neutrophils are fundamental in recruiting mononuclear leukocytes to mount an immune response at the injury site. Neutrophils exert significant pro-inflammatory effects through the release of cytokines and chemokines, degranulation and release of myeloperoxidase (MPO) and neutrophil elastase (NE), and the production of large DNA-based networks called neutrophil extracellular traps (NETs). Neutrophils are initially recruited and activated by cytokines and pathogen- and damage-associated molecular patterns, but little is known about how the physicochemical composition of the biomaterial affects their activation. This study aimed to understand how ablating neutrophil mediators (MPO, NE, NETs) affected macrophage phenotype in vitro and osseointegration in vivo. We discovered that NET formation is a crucial mediator of pro-inflammatory macrophage activation, and inhibition of NET formation significantly suppresses macrophage pro-inflammatory phenotype. Furthermore, reducing NET formation accelerated the inflammatory phase of healing and produced greater bone formation around the implanted biomaterial, suggesting that NETs are essential regulators of biomaterial integration. Our findings emphasize the importance of the neutrophil response to implanted biomaterials and highlight innate immune cells' regulation and amplification signaling during the initiation and resolution of the inflammatory phase of biomaterial integration. STATEMENT OF SIGNIFICANCE: Neutrophils are the most abundant immune cells in blood and are the first to be recruited to the injury/implantation site where they exert significant pro-inflammatory effects. This study aimed to understand how ablating neutrophil mediators affected macrophage phenotype in vitro and bone apposition in vivo. We found that NET formation is a crucial mediator of pro-inflammatory macrophage activation. Reducing NET formation accelerated the inflammatory phase of healing and produced greater appositional bone formation around the implanted biomaterial, suggesting that NETs are essential regulators of biomaterial integration.

S100A9 is indispensable for survival of pneumococcal pneumonia in mice.

S100A8/A9 has important immunomodulatory roles in antibacterial defense, but its relevance in focal pneumonia caused by Streptococcus pneumoniae (S. pneumoniae) is understudied. We show that S100A9 was significantly increased in BAL fluids of patients with bacterial but not viral pneumonia and correlated with procalcitonin and sequential organ failure assessment scores. Mice deficient in S100A9 exhibited drastically elevated Zn2+ levels in lungs, which led to bacterial outgrowth and significantly reduced survival. In addition, reduced survival of S100A9 KO mice was characterized by excessive release of neutrophil elastase, which resulted in degradation of opsonophagocytically important collectins surfactant proteins A and D. All of these features were attenuated in S. pneumoniae-challenged chimeric WT→S100A9 KO mice. Similarly, therapy of S. pneumoniae-infected S100A9 KO mice with a mutant S100A8/A9 protein showing increased half-life significantly decreased lung bacterial loads and lung injury. Collectively, S100A9 controls central antibacterial immune mechanisms of the lung with essential relevance to survival of pneumococcal pneumonia. Moreover, S100A9 appears to be a promising biomarker to distinguish patients with bacterial from those with viral pneumonia. Trial registration: Clinical Trials register (DRKS00000620).

Derangement in Homeostasis of Neutrophil Elastase and its Inhibitory Systems in Ischemic Stroke Patients

Neutrophils are the first to infiltrate ischemic brain regions causing the release of Neutrophil Elastase (NE), a pro-inflammatory proteinase. The activity of NE is well regulated by endogenous inhibitors alpha1-antitrypsin (α1-AT) and alpha2-macroglobulin (α2-MG). The physiological balance of elastase and anti-elastase factors is essential to maintain the normal integrity of tissues and an imbalance has been implicated in the pathogenesis of several acute and chronic inflammatory diseases. The present study was designed to determine the plasma levels of NE, α1-AT, α2-MG, and NE–α1-AT complex to evaluate their role in inflammatory processes of ischemic stroke. The effect of homocysteine on the release of elastase from neutrophils was also studied. The study involved a total of 100 subjects (controls =60 and patients=40). Significantly higher mean elastase activity and lower α1-AT levels were observed in ischemic stroke patients than in controls. NE- α1-AT complex and α2-MG levels were significantly increased in the patient group. The in vitro study indicated homocysteine induced release of elastase from neutrophils. In conclusion, homeostasis of NE and its endogenous inhibitors is deranged in patients suggestive of their role in the pathogenesis of ischemic stroke through exacerbating inflammatory and coagulation processes.

Elevation of neutrophil-derived factors in patients after multiple trauma.

Trauma represents one of the leading causes of death worldwide. Traumatic injuries elicit a dynamic inflammatory response with systemic release of inflammatory cytokines. Disbalance of this response can lead to systemic inflammatory response syndrome or compensatory anti-inflammatory response syndrome. As neutrophils play a major role in innate immune defence and are crucial in the injury-induced immunological response, we aimed to investigate systemic neutrophil-derived immunomodulators in trauma patients. Therefore, serum levels of neutrophil elastase (NE), myeloperoxidase (MPO) and citrullinated histone H3 (CitH3) were quantified in patients with injury severity scores above 15. Additionally, leukocyte, platelet, fibrinogen and CRP levels were assessed. Lastly, we analysed the association of neutrophil-derived factors with clinical severity scoring systems. Although the release of MPO, NE and CitH3 was not predictive of mortality, we found a remarkable increase in MPO and NE in trauma patients as compared with healthy controls. We also found significantly increased levels of MPO and NE on Days 1 and 5 after initial trauma in critically injured patients. Taken together, our data suggest a role for neutrophil activation in trauma. Targeting exacerbated neutrophil activation might represent a new therapeutic option for critically injured patients.

Biological mechanisms of infection resistance in tissue engineered blood vessels compared to synthetic expanded polytetrafluoroethylene grafts

ObjectiveSynthetic expanded polytetrafluoroethylene (ePTFE) grafts are known to be susceptible to bacterial infection. Results from preclinical and clinical studies of bioengineered human acellular vessels (HAVs) have shown relatively low rates of infection. This study evaluates the interactions of human neutrophils and bacteria with ePTFE and HAV vascular conduits to determine whether there is a correlation between neutrophil-conduit interactions and observed differences of their infectivity in vivo. MethodsA phase III comparative clinical study between investigational HAVs (n = 177) and commercial ePTFE grafts (n = 178) used for hemodialysis access (ClinicalTrials.gov Identifier: NCT02644941) was evaluated for conduit infection rates followed by histological analyses of HAV and ePTFE tissue explants. The clinical histopathology of HAV and ePTFE conduits reported to be infected was compared with immunohistochemistry of explanted materials from a preclinical model of bacterial contamination. Mechanistic in vitro studies were then conducted using isolated human neutrophils seeded directly onto HAV and ePTFE materials to analyze neutrophil viability, morphology, and function. ResultsClinical trial results showed that the HAV had a significantly lower (0.93%; P = .0413) infection rate than that of ePTFE (4.54%). Histological analysis of sections from infected grafts explanted approximately 1 year after implantation revealed gram-positive bacteria near cannulation sites. Immunohistochemistry of HAV and ePTFE implanted in a well-controlled rodent infection model suggested that the ePTFE matrix permitted bacterial infiltration and colonization but may be inaccessible to neutrophils. In the same model, the HAV showed host recellularization and lacked detectable bacteria at the 2-week explant. In vitro results demonstrated that the viability of human neutrophils decreased significantly upon exposure to ePTFE, which was associated with neutrophil elastase release in the absence of bacteria. In contrast, neutrophils exposed to the HAV material retained high viability and native morphology. Cocultures of neutrophils and Staphylococcus aureus on the conduit materials demonstrated that neutrophils were more effective at ensnaring and degrading bacteria on the HAV than on ePTFE. ConclusionsThe HAV material seems to demonstrate a resistance to bacterial infection. This infection resistance is likely due to the HAV's native-like material composition, which may be more biocompatible with host neutrophils than synthetic vascular graft material.

Moving into the red - a near infra-red optical probe for analysis of human neutrophil elastase in activated neutrophils and neutrophil extracellular traps.

Neutrophils are the first immune cells recruited for defence against invading pathogens; however, their dysregulated activation and subsequent release of the enzyme human neutrophil elastase is associated with several, inflammation-based, diseases. Herein, we describe a FRET-based, tri-branched (one quencher, three fluorophores) near infrared probe that provides an intense OFF/ON amplified fluorescence signal for specific detection of human neutrophil elastase. The probe allowed selective detection of activated neutrophils and labelling of neutrophil extracellular traps.

Tanshinone IIA Improves Acute Gouty Arthritis in Rats through Regulating Neutrophil Activation and the NLRP3 Inflammasome.

To investigate the prevention and treatment effect of tanshinone IIA (TIIA) on acute gouty arthritis (AGA) and its mechanism. The anti-AGA effect of TIIA was observed in vivo and in vitro. Neutrophils were isolated from the abdominal cavity of mice, and the anti-AGA effect of TIIA was investigated in a rat model of MSU-induced AGA. The pathological changes of the ankle joint tissues were assessed by H&E. Cytokine and chemokine expression were determined by ELISA and RT-qPCR. The NLRP3 inflammasome pathway protein levels in the ankle joint tissues were evaluated via western blotting. Neutrophil migration was evaluated in air pouch and transwell assays. Immunohistochemistry and immunofluorescence analysis evaluate the release of myeloperoxidase (MPO), neutrophil elastase (NE), and citrullination of histone H3 (CitH3). Beclin-1 and LC3B expressions were determined using western blotting and immunofluorescence. Key Findings. Treatment with TIIA alleviated synovial hyperplasia and neutrophil infiltration, regulated cytokine and chemokine expressions, and inhibited NLRP3 activation in AGA rats, neutrophil migration, MPO, NE, and CitH3 expression, and LC3B and Beclin-1 protein expression. These results demonstrate that TIIA can effectively enhance AGA by focusing on the neutrophils and NLRP3 inflammasome, demonstrating that TIIA may act as a potential helpful agent for AGA.

Fibrotic Lung Disease Alters Neutrophil Trafficking and Promotes Neutrophil Elastase and Extracellular Trap Release.

Idiopathic pulmonary fibrosis (IPF) is a progressive, irreversible disease characterized by collagen deposition within the interstitium of the lung. This impairs gas exchange and results in eventual respiratory failure. Clinical studies show a correlation between elevated neutrophil numbers and IPF disease progression; however, the mechanistic roles neutrophils play in this disease are not well described. In the present study, we describe alterations to the trafficking and function of neutrophils after the development of fibrosis. We observed increased numbers of total and aged neutrophils in peripheral tissues of fibrotic mice. This appeared to be driven by an upregulation of neutrophil chemokine Cxcl2 by lung cells. In addition, neutrophil recruitment back to the bone marrow for clearance appeared to be impaired, because we saw decreased aged neutrophils in the bone marrow of fibrotic mice. Neutrophils in fibrosis were activated, because ex vivo assays showed increased elastase and extracellular trap release by neutrophils from fibrotic mice. This likely mediated disease exacerbation, because mice exhibiting a progressive disease phenotype with greater weight loss and mortality had more activated neutrophils and increased levels of extracellular DNA present in their lungs than did mice with a nonprogressive disease phenotype. These findings further our understanding of the dynamics of neutrophil populations and their trafficking in progressive fibrotic lung disease and may help inform treatments targeting neutrophil function for patients with IPF experiencing disease exacerbation in the future.

Research progress of effect of neutrophil elastase and its inhibitors in sepsis

Sepsis is a clinical syndrome of life-threatening organ dysfunction caused by infection. When an infection occurs, as the first line of defense of the body's immune system, neutrophils are first recruited to the site of infection to capture and kill pathogens by releasing neutrophil elastase (NE). However, a large amount of NE release will injury the surrounding normal tissues and induce organ dysfunction or failure. NE inhibitors can inhibit NE activity and reduce inflammatory response, which may be a promising drug for the treatment of sepsis. Currently, a variety of NE inhibitors have been developed and reported, but there is no systematic overview of their characteristics, and the role and underlying mechanisms of NE and related inhibitors in sepsis have not been thoroughly discussed. This article will make a review in this regard, in order to elucidate the effect of NE and its inhibitors in sepsis.

Doxorubicin-induced cardiotoxicity is mediated by neutrophils through release of neutrophil elastase.

The mechanisms by which Doxorubicin (Dox) causes acute and late cardiotoxicity are not completely understood. One understudied area is the innate immune response, and in particular the role of neutrophils in Dox-induced cardiotoxicity. Here, using echocardiography, flow cytometry and immunofluorescence staining, we demonstrated increased infiltration of neutrophils that correlated with decreased heart function, disruption of vascular structures and increased collagen deposition in the heart after Dox treatment. Depleting neutrophils protected the heart from Dox-induced cardiotoxicity and changes in vascular structure. Furthermore, our data using neutrophil elastase (NE) knock-out mice and the NE inhibitor AZD9668 suggest that neutrophils cause this damage by releasing NE and that inhibiting NE can prevent Dox-induced cardiotoxicity. This work shows the role of neutrophils and NE in Doxorubicin-induced cardiotoxicity for the first time and suggests a new possible therapeutic intervention.