Targeted Library of Phosphonic-Type Inhibitors of Human Neutrophil Elastase.

Despite many years of research, human neutrophil elastase (HNE) still remains an area of interest for many researchers. This multifunctional representative of neutrophil serine proteases is one of the most destructive enzymes found in the human body which can degrade most of the extracellular matrix. Overexpression or dysregulation of HNE may lead to the development of several inflammatory diseases. Previously, we presented the HNE inhibitor with kinact/KI value over 2,000,000 [M-1s-1]. In order to optimize its structure, over 100 novel tripeptidyl derivatives of α-aminoalkylphosphonate diaryl esters were synthesized, and their activity toward HNE was checked. To confirm the selectivity of the resultant compounds, several of the most active were additionally checked against the two other neutrophil proteases: proteinase 3 and cathepsin G. The developed modifications allowed us to obtain a compound with significantly increased inhibitory activity against human neutrophil elastase with high selectivity toward cathepsin G, but none toward proteinase 3.

Resveratrol Attenuates Rheumatoid Arthritis Induce Neutrophil Extracellular Traps via TLR-4 Mediated Inflammation in C57BL/6 Mice.

The objective of this study was to evaluate whether RSV inhibits neutrophil extracellular traps (NETs) that induce joint hyperalgesia in C57BL/6 mice after adjuvant-induced arthritis. A subplantar injection of Freund's complete adjuvant was administered to C57BL/6 mice on day 0 for immunization in the AIA model. Resveratrol (RSV, 25 mg/kg) was administered intraperitoneally once daily starting on day 22 and continuing for two weeks. The effects of mechanical hyperalgesia and edema formation have been assessed in addition to histopathological scoring. Mice were sacrificed on day 35 to determine cytokine levels and PADI4 and COX-2 expression levels. ELISA was used to quantify neutrophil extracellular traps (NETs) along with neutrophil elastase-DNA and myeloperoxidase-DNA complexes in neutrophils. An immunohistochemical stain was performed on knee joints to determine the presence of nuclear factor kappa B p65 (NF-kappaB p65). AIA mice were found to have higher levels of NET in joints and their joint cells demonstrated an increased expression of the PADI4 gene. Treatment with RSV in AIA mice (25 mg/kg, i.p.) significantly (P<0.05) inhibited joint hyperalgesia, resulting in a significant increase in mechanical threshold, a decrease in articular edema, a decrease in the production of inflammatory cytokines, increased COX-2 expression, and a decrease in the immunostaining of NF-kappaB. Furthermore, treatment with RSV significantly reduced the amount of neutrophil elastase (NE)-DNA and MPO-DNA complexes, which were used as indicators of NET formation (P<0.05). This study indicates that RSV reduces NET production and hyperalgesia by reducing inflammation mediated by PADI4 and COX-2. According to these data, NETs contribute to joint pain and resveratrol can be used to treat pain in RA through this pathway.

Galectin-3 levels in children with cystic fibrosis.

Cystic fibrosis (CF) is a multisystemic disease in which airway obstruction, infection, and inflammation play a critical role in the pathogenesis and progression of CF lung disease. The carbohydrate-binding protein Galectin-3 is increased in several inflammatory and fibrotic diseases and has recently been forwarded as a biomarker in these diseases. We aimed to define the role of serum Galectin-3 in children with CF by comparison with healthy subjects. This is a cross-sectional, case-control study. 143 CF and 30 healthy subjects were enrolled in the study. Peripheral blood and sputum concentrations of Galectins-3, interleukin (IL)-17A, IL-8, and neutrophil elastase (NE) were determined with commercial ELISA kits. There was no significant difference between the groups in age and gender (p = 0.592, p = 0.613, respectively). Serum Galectin-3 and NE concentrations were higher in the patient group than in healthy controls (p = 0.002, p < 0.001, respectively). There were no significant differences between groups according to IL-17A and IL-8 concentrations. Serum Galectin-3 was correlated with age (r = 0.289, p < 0.001) and body mass index (BMI) (r = 0.493, p < 0.001) in children with CF. Sputum Galectin-3 levels are negatively correlated with percent predictive forced expiratory volume in 1 s (FEV1) (r = - 0.297, p = 0.029), FEV1 z-score, (r = - 0.316, p = 0.020), percent predictive forced vital capacity (FVC) (r = - 0.347, p = 0.010), and FVC z-score (r = - 0.373, p = 0.006).   Conclusion: The study shows that serum Galectin-3 levels increased in clinically stable CF patients, and serum Galectin-3 response may depend on age, gender, and BMI. The sputum Galectin-3 was found to be negatively correlated with patients' lung functions. What is known: • Galectin-3 is a key regulator of chronic inflammation in the lung, liver, kidney, and tumor microenvironment. What is new: • Children with cystic fibrosis (CF) have higher serum Galectin-3 concentrations than healthy children. • Serum Galectin-3 expression influenced by age, BMI, and gender in children with CF.

Safety, tolerability, pharmacokinetics and neutrophil elastase inhibitory effects of Sivelestat: A randomized, double-blind, placebo-controlled single- and multiple-dose escalation study in Chinese healthy subjects.

BACKGROUND AND OBJECTIVE: Neutrophil elastase has been identified as a potential therapeutic target for acute lung injury or acute respiratory distress syndrome, and Sivelestat is a selective, reversible and competitive neutrophil elastase inhibitor. This study was designed to investigate the safety, tolerability, pharmacokinetics and neutrophil elastase inhibitory effects of Sivelestat in healthy Chinese subjects. METHODS: A randomized, double-blind, placebo-controlled single- and multiple-dose escalation clinical trial was carried out. Briefly, healthy volunteers in twelve cohorts with 8 per cohort received 1.0-20.2 mg/kg/h Sivelestat or placebo in an intravenous infusion manner for two hours, and healthy volunteers in four cohorts received two hours intravenous infusion of 2.0-5.0 mg/kg/h Sivelestat or placebo with an interval of twelve hours for seven times. The safety and tolerability were evaluated and serial blood samples were collected for pharmacokinetics and neutrophil elastase inhibitory effects analysis at the specified time-point. RESULTS: A total of 128 subjects were enrolled and all participants completed the study except one. Sivelestat exhibited satisfactory safety and tolerability up to 20.2 mg/kg/h in single-dose cohorts and 5.0 mg/kg/h in multiple-dose cohorts. Even so, more attention should be paid to the safety risks when using high doses. The Cmax and AUC of Sivelestat increased in a dose dependent manner, and Tmax was similar for different dose cohorts. In multiple-dose cohorts, the plasma concentrations reached steady state 48 h after first administration and the accumulation of Cmax and AUC was not obvious. Furthermore, the Cmin_ss of 5.0 mg/kg/h dose cohort could meet the needs of clinical treatment. For some reason, the pharmacodynamics data revealed that the inhibitory effect of Sivelestat on neutrophil elastase content in healthy subjects was inconclusive. CONCLUSION: Sivelestat was safe and well tolerated with appropriate pharmacokinetic parameters, which provided support for more diverse dosing regimen in clinical application. CLINICAL TRIAL REGISTRATION: www.chinadrugtrials.org.cn identifier is CTR20210072.

Elevated first-trimester neutrophil elastase and proteinase 3 increase the risk of gestational diabetes mellitus and adverse fetal outcomes.

BACKGROUND: Chronic inflammation plays a vital role in the development of gestational diabetes mellitus (GDM). Studies in mouse models show that neutrophil serine proteases (NSPs), neutrophil elastase (NE) and proteinase-3 (PR3) are important drivers of chronic inflammation with consequent metabolic disturbances. This study evaluated the association of NE and PR3 with GDM development and adverse fetal outcomes. METHOD(S): This was a prospective cohort study. Serum PR3 and NE concentration was measured in all enrolled pregnant women in the first and the second trimester to determine the connection between NSPs and GDM and adverse fetal outcomes. Logistic regression, spline regression and linear regression analyses were applied to investigate the association of NE or PR3 with GDM development and adverse fetal outcomes. The concentration of NE and PR3 in placental biopsies was evaluated by semi-quantitative analysis of immunohistochemistry staining. RESULT(S): NE or PR3 concentration in the first trimester, rather than the second, increased more significantly in women with GDM than in those without, regardless of pre-pregnancy body mass index and age. There was a stepwise increase in GDM occurrence as well as comprehensive adverse fetal outcomes across tertiles of NE and PR3. NE and PR3 were positively associated with neutrophil count, pre-pregnancy BMI, plasma glucose level and newborn weight. Logistic regression revealed NE or PR3 to be independent risk factors for the development of GDM and comprehensive adverse fetal outcomes. Spline regression showed a significant increased risk of GDM occurrence and comprehensive adverse fetal outcomes when serum NE concentration exceeded 417.60 ng/mL and a similar result for PR3 and GDM occurrence when the latter exceeded 88.52 ng/mL. Immunohistochemistry data confirmed that enriched NE and PR3 content in placental tissue may have contributed to the development of GDM. CONCLUSION(S): This work demonstrates that excessive first-trimester NE and PR3 increase the risk of GDM development and comprehensive adverse fetal outcomes.

Changes in Circulating Acylated Ghrelin and Neutrophil Elastase in Diabetic Retinopathy.

Background and Objectives: The role and the levels of ghrelin in diabetes-induced retinal damage have not yet been explored. The present study aimed to measure the serum levels of total ghrelin (TG), and its acylated (AG) and des-acylated (DAG) forms in patients with the two stages of diabetic retinopathy (DR), non-proliferative (NPDR) and proliferative (PDR). Moreover, the correlation between serum ghrelin and neutrophil elastase (NE) levels was investigated. Materials and Methods: The serum markers were determined via enzyme-linked immunosorbent assays in 12 non-diabetic subjects (CTRL), 15 diabetic patients without DR (Diabetic), 15 patients with NPDR, and 15 patients with PDR. Results: TG and AG serum levels were significantly decreased in Diabetic (respectively, p < 0.05 and p < 0.01 vs. CTRL), NPDR (p < 0.01 vs. Diabetic), and in PDR patients (p < 0.01 vs. NPDR). AG serum levels were inversely associated with DR abnormalities (microhemorrhages, microaneurysms, and exudates) progression (r = -0.83, p < 0.01), serum neutrophil percentage (r = -0.74, p < 0.01), and serum NE levels (r = -0.73, p < 0.01). The latter were significantly increased in the Diabetic (p < 0.05 vs. CTRL), NPDR (p < 0.01 vs. Diabetic), and PDR (p < 0.01 vs. PDR) groups. Conclusions: The two DR stages were characterized by decreased AG and increased NE levels. In particular, serum AG levels were lower in PDR compared to NPDR patients, and serum NE levels were higher in the PDR vs. the NPDR group. Together with the greater presence of retinal abnormalities, this could underline a distinctive role of AG in PDR compared to NPDR.

Position-specific N- and O-glycosylation of the reactive center loop impacts neutrophil elastase-mediated proteolysis of corticosteroid-binding globulin.

Corticosteroid-binding globulin (CBG) delivers anti-inflammatory cortisol to inflamed tissues through proteolysis of an exposed reactive center loop (RCL) by neutrophil elastase (NE). We previously demonstrated that RCL-localized Asn347-linked N-glycans impact NE proteolysis, but a comprehensive structure-function characterization of the RCL glycosylation is still required to better understand CBG glycobiology. Herein, we first performed RCL-centric glycoprofiling of serum-derived CBG to elucidate the Asn347-glycans and then used molecular dynamics simulations to study their impact on NE proteolysis. Importantly, we also identified O-glycosylation (di/sialyl T) across four RCL sites (Thr338/Thr342/Thr345/Ser350) of serum CBG close to the NE-targeted Val344-Thr345 cleavage site. A restricted N- and O-glycan co-occurrence pattern on the RCL involving exclusively Asn347 and Thr338 glycosylation was experimentally observed and supported in silico by modeling of a CBG-GalNAc-transferase (GalNAc-T) complex with various RCL glycans. GalNAc-T2 and GalNAc-T3 abundantly expressed by liver and gall bladder, respectively, showed in vitro a capacity to transfer GalNAc (Tn) to multiple RCL sites suggesting their involvement in RCL O-glycosylation. Recombinant CBG was then used to determine roles of RCL O-glycosylation through longitudinal NE-centric proteolysis experiments, which demonstrated that both sialoglycans (disialyl T) and asialoglycans (T) decorating Thr345 inhibit NE proteolysis. Synthetic RCL O-glycopeptides expanded on these findings by showing that Thr345-Tn and Thr342-Tn confer strong and moderate protection against NE cleavage, respectively. Molecular dynamics substantiated that short Thr345-linked O-glycans abrogate NE interactions. In conclusion, we report on biologically relevant CBG RCL glycosylation events, which improve our understanding of mechanisms governing cortisol delivery to inflamed tissues.

An elastase-inhibiting, plaque-targeting and neutrophil-hitchhiking liposome against atherosclerosis.

Neutrophil extracellular traps (NETs) play a crucial role in the formation of vulnerable plaques and the development of atherosclerosis. Alleviating the pathological process of atherosclerosis by efficiently targeting neutrophils and inhibiting the activity of neutrophil elastase to inhibit NETs is relatively unexplored and is considered a novel therapeutic strategy with clinical significance. Sivelestat (SVT) is a second-generation competitive inhibitor of neutrophil elastase with high specificity. However, therapeutic effect of SVT on atherosclerosis is restricted because of the poor half-life and the lack of specific targeting. In this study, we construct a plaque-targeting and neutrophil-hitchhiking liposome (cRGD-SVT-Lipo) to improve the efficacy of SVT in vivo by modifying the cRGD peptide onto SVT loaded liposome, which was based on the interaction between cRGD peptide and integrin ανß3 on the surface of cells in blood and plaque, including epithelial cell, macrophage and neutrophils. The cRGD-SVT-Lipo could actively tend to or hitchhike neutrophils in situ to reach atherosclerotic plaque, which resulted in enhanced atherosclerotic plaque delivery. The cRGD-SVT-Lipo could also reduce plaque area, stabilize plaque, and ultimately alleviate atherosclerosis progression through efficiently inhibiting the activity of neutrophil elastase in atherosclerotic plaque. Therefore, this study provides a basis and targeting strategy for the treatment of neutrophil-related diseases. STATEMENT OF SIGNIFICANCE: Neutrophil extracellular traps (NETs)-inhibiting is a prospective therapeutic approach for atherosclerosis but has received little attention. The NETs can be inhibited by elastase-restraining. In this work, an intriguing system that delivers Sivelestat (SVT), a predominantly used neutrophil elastase inhibitor with poor targeting capability, is designed to provide the drug with plaque-targeting and neutrophil-hitchhiking capability. The result suggests that this system can effectively hinder the formation of NETs and delay the progression of atherosclerosis.

Human neutrophil elastase inhibitors: Classification, biological-synthetic sources and their relevance in related diseases.

BACKGROUND: Human neutrophil elastase is a multifunctional protease enzyme whose function is to break the bonds of proteins and degrade them to polypeptides or amino acids. In addition, it plays an essential role in the immune mechanism against bacterial infections and represents a key mediator in tissue remodeling and inflammation. However, when the extracellular release of this enzyme is dysregulated in response to low levels of its physiological inhibitors, it ultimately leads to the degradation of proteins, in particular elastin, as well as other components of the extracellular matrix, producing injury to epithelial cells, which can promote sustained inflammation and affect the innate immune system, and, therefore, be the basis for the development of severe inflammatory diseases, especially those associated with the cardiopulmonary system. OBJECTIVE: This review aims to provide an update on the elastase inhibitory properties of several molecules, either synthetic or biological sources, as well as their classification and relevance in related pathologies since a clear understanding of the function of these molecules with the inhibitory capacity of this protease can provide valuable information for the development of pharmacological therapies that manage to modify the prognosis and survival of various inflammatory diseases. METHODS: Collected data from scientific databases, including PubMed, Google Scholar, Science Direct, Nature, Wiley, Scopus, and Scielo. Articles published in any country and language were included. RESULTS: We reviewed and included 132 articles conceptualizing neutrophil elastase activity and known inhibitors. CONCLUSION: Understanding the mechanism of action of elastase inhibitors based on particular aspects such as their kinetic behavior, structure-function relationship, chemical properties, origin, pharmacodynamics, and experimental progress has allowed for a broad classification of HNE inhibitors.

Botulinum toxin type A ameliorates rat dorsal root ganglia neuron pyroptosis in postherpetic neuralgia by upregulating cathelicidin antimicrobial peptide to inhibit neutrophil elastase.

Botulinum toxin type A (BoNT/A) has exhibited efficacy in postherpetic neuralgia (PHN) treatment, and this study aims to uncover its underlying mechanisms. Resiniferatoxin (RTX)-induced PHN rats were given BoNT/A. Rat postoperative pain behaviors were assessed by Von Frey test. Cleaved-synaptosomal protein 25 kDa (cl-SNAP-25) or cathelicidin antimicrobial peptide (CAMP) expression in rat dorsal root ganglia (DRG) was detected by immunofluorescence or immunohistochemistry. Healthy rat-derived DRG neurons were transfected, incubated with lipopolysaccharides (LPS)/adenosine 5'-triphosphate (ATP) to stimulate pyroptosis and treated with BoNT/A. The CCK-8, Western blot, ELISA, and qRT-PCR were used to assess the viability, levels of pyroptosis-related proteins proinflammatory cytokine levels, as well as CAMP and ELANE mRNA levels. BoNT/A (30 U/kg) promoted cl-SNAP-25 expression in rat DRG and reversed RTX-induced decrease of rat paw withdrawal thresholds and CAMP expression and increase of pyroptosis-associated protein and inflammatory factor expression in rat DRG. CAMP interacted with ELANE in rat DRG neurons. BoNT/A attenuated LPS/ATP-stimulated inhibition of viability and CAMP expression and upregulation of inflammatory mediators, pyroptosis-related proteins, and ELANE expression in rat DRG neurons, which was counteracted by CAMP silencing. However, ELANE knockdown offset the effect of CAMP silencing in LPS/ATP/BoNT/A-treated rat DRG neurons. On the whole, BoNT/A alleviates rat DRG neuron pyroptosis during PHN by upregulating CAMP to inhibit ELANE.

Measuring the Concentration of Active Alpha-1 Antitrypsin in Serum.

Active Alpha-1 antitrypsin (AAT) circulates in blood in two isoforms in dynamic equilibrium: 1) native AAT, which binds irreversibly to neutrophil elastase, and 2) thiol-modified AAT, which binds reversibly to neutrophil elastase, either of which can be inactivated by oxidation, cleavage, covalent binding, and bound antibodies. Anti-AAT antibodies used for detecting AAT in plasma bind to active and inactive protein, thereby overestimating the concentration of functional protein. Active AAT can be quantitated by measuring its inhibition of active-site titrated elastase. A method for measuring active AAT in the presence of competing proteinase inhibitors in plasma has been developed and is described herein.

[Features of inflammatory reactions in patients with juvenile depression with a clinically high risk of psychosis]. / Osobennosti vospalitel'nykh reaktsii u patsientov s yunosheskimi depressiyami s klinicheski vysokim riskom psikhoza.

OBJECTIVE: To determine the levels of pro-inflammatory and anti-inflammatory cytokines and inflammatory markers such as C-reactive protein, leukocyte elastase, α1-proteinase inhibitor, autoantibodies to neuroantigens in the blood of patients with adolescent depression with clinical high risk for psychosis (CHR-P) and to study the relation of these biological markers to the features of psychopathological symptomatology of the patients. MATERIAL AND METHODS: Eighty young adults, aged 16-24 years, with the first depressive episode (F32.1-2, F32.38, F32.8) were studied. Based on the presence of attenuated positive symptoms in the structure of depression, all patients were divided into two groups: with CHR-P (clinical group, n=58) and without CHR-P (comparative group, n=22). The HDRS-21, SOPS, and SANS were used for psychometric assessment of the patients. Serum levels of cytokines TNF-α, IL-6, IL-8, IL-10, and concentration of C-reactive protein (CRP) were determined. Leukocyte elastase (LE) activity, α1-proteinase inhibitor (α1-PI) activity, and plasma levels of autoantibodies to S100B protein and myelin basic protein (MBP) were assessed. RESULTS: Both groups of patients were characterized by the high levels of inflammation as assessed by LE (250.5 (226.2-280.8) nmol/min·ml vs 248.3 (226.8-284.5) nmol/min·ml) and α1-PI activity (44.4 (37.5-50.1) IE/ml vs 45.2 (36.4-49.9) IE/ml). Higher levels (p<0.05) of IL-6 (1.22 (0.64-2.2) pg/ml), CRP (0.93 (0.18-3.18) mg/l), and TNF-α/IL-10 (0.34 (0.2-0.47)) were detected in the group with CHR-P. This group was also characterized by higher levels of antibodies to the S100B protein 0.78 (0.69-0.84 units of opt.density) compared with the group without CRH-P (p<0.05). In each clinical group, different correlations between clinical, psychometric and biological parameters were revealed. CONCLUSIONS: The results confirm the involvement of inflammation in the development of depression in youth and indicate a different role of the inflammatory markers analyzed in the formation of CHR-P. The differences in the spectrum of inflammatory markers in depressed patients suggest a more pronounced pro-inflammatory potential in the group with CHR-P.

Lead activates neutrophil degranulation to induce early myocardial injury in mice.

Lead (Pb) is a pervasive toxic metal contaminant associated with a high risk of myocardial injury. However, the precise mechanism underlying Pb-induced myocardial injury has yet to be fully elucidated. In this study, a murine model of Pb exposure (0, 1, 5, and 10 mg/kg) was employed to investigate the involvement of neutrophil degranulation in the induction of myocardial injury. Notably, serum levels of cardiac troponin I (cTnI) and creatine kinase-MB (CK-MB) increased significantly in Pb-exposed mice, whereas cTnI levels in cardiomyocytes decreased, suggesting that Pb exposure may cause early myocardial injury. Moreover, Pb exposure was found to promote neutrophil degranulation, as evidenced by elevated myeloperoxidase (MPO) and neutrophil elastase (NE) concentrations in both the serum of Pb-exposed workers and Pb-exposed mice, as well as the extracellular supernatant of neutrophils following exposure. However, we found that serum level of cTnI enhanced by Pb exposure is associated with increased NE levels in the serum, but not with MPO levels. Upon treatment with NE inhibitor (sivelestat), the serum level of cTnI markedly reduced in Pb-exposed mice, we found that early myocardial injury is associated with NE levels in the serum. At the molecular level, western blotting analysis revealed an upregulation of ERK1/2 expression in vitro following Pb exposure, suggesting that the activation of the ERK1/2 signaling pathway may underlie the participation of neutrophil degranulation in Pb-induced myocardial injury. In summary, our findings demonstrate that Pb exposure can initiate early myocardial injury by promoting the neutrophil degranulation process, thereby highlighting the potential role of this process in the pathogenesis of Pb-associated myocardial injury.

Screening for ELANE, HAX1 and GFI1 gene mutations in children with neutropenia and clinical characterization of two novel mutations in ELANE gene.

BACKGROUND: Congenital neutropenia is a rare disease. Recurrent infections since young age are the presentation. The most common mutation causing severe congenital neutropenia (SCN) and cyclic neutropenia (CyN) is the ELANE gene. The objectives of this study were to screen the three common genetic mutations of ELANE, HAX1 and GFI1 in children with chronic neutropenia and to describe the clinical characteristics of children who had the mutations. METHODS: Infants having ANC < 1,000/cu mm or children aged > 1 year having ANC < 1,500/cu mm at least 3 times in 3 months were enrolled in the study. Patients who had acquired neutropenia due to infection, immune deficiency, or drugs were excluded. The ELANE gene was first studied; and if mutations were not identified, the HAX1 and GFI1 genes were further examined. RESULTS: A total of 60 patients were enrolled in the study. The median (range) age, ratio of female to male, ANC, and last follow-up age were 9.2 (0.5-45.2) months, 1:1.2, 248 (0-1,101) /cu mm, and 19.9 (3.5-202.3) months, respectively. Infections were noted in 67.3% of all patients. ELANE gene mutation was found in only four patients (6.7%), and the rest (56 patients) showed no mutations in the HAX1 and GFI1 genes. In patients without mutations, 66.0% had normal ANC during the follow-up, with a median (range) age for normal ANC of 19.8 (4.0-60.0) months. Two novel mutations p. Ala79del (c.234_236del) and p. Val197GlufsTer18 (c.589_590insAGGCCGGC) were identified, and they respectively cause SCN and CyN. Patients with the two novel mutations presented with several episodes of infection, including pneumonia, sepsis, abscess, otitis media, and gum infection. CONCLUSION: The genetic screening for ELANE, HAX1, and GFI1 gene mutations in 60 patients with chronic neutropenia could identify four patients (6.7%) with ELANE gene mutation and two novel mutations, p. Ala79del in exon 3 and p. Val197GlufsTer18 in exon 4 causing SCN; and CyN, respectively.

Neutrophil elastase activates the release of extracellular traps from COPD blood monocyte-derived macrophages.

Neutrophil elastase (NE), a major inflammatory mediator in chronic obstructive pulmonary disease (COPD) airways, impairs macrophage function, contributing to persistence of airway inflammation. We hypothesized that NE activates a novel mechanism of macrophage-induced inflammation: release of macrophage extracellular traps (METs). The METs are composed of extracellular DNA decorated with granule proteinases and oxidants and may trigger persistent airway inflammation in COPD. To test the hypothesis, human blood monocytes were isolated from whole blood of subjects with COPD recruited following informed written consent. Patient demographics and clinical data were collected. Cells were cultured in media with GM-CSF to differentiate into blood monocyte derived macrophages (BMDMs). The BMDMs were treated with FITC-NE and unlabeled NE to determine intracellular localization by confocal microscopy and intracellular proteinase activity by DQ-Elastin assay. After NE exposure, released extracellular traps were quantified by abundance of extracellular DNA in conditioned media using the Pico Green assay. BMDM cell lysates were analyzed by Western analysis for proteolytic degradation of histone H3 or H4 or upregulation of peptidyl arginine deiminase (PAD) 2 and 4, two potential mechanisms to mediate extracellular trap DNA release. We observed that NE was taken up by COPD BMDM, localized to the cytosol and nucleus, and retained proteinase activity in the cell. NE induced MET release at doses as low as 50 nM. NE treatment caused histone H3 clipping but no effect on histone H4 nor PAD 2 or 4 abundance or activity. In summary, NE activated COPD MET release by clipping histone H3, a prerequisite for chromatin decondensation.