Neutrophil Elastase Activity Research Articles

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 ανβ3 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 significanceNeutrophil 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.

A non-peptide-based chemiluminescent probe for sensitively visualizing neutrophil elastase activity in vivo

As a prototypical serine protease, neutrophil elastase (NE) plays a pivotal role in the regulation of inflammation, and its dysregulated activation is closely associated with organ damage related to inflammation. Despite the existence of several NE-responsive fluorescent probes, they are still susceptible to interference from auto-fluorescence caused by real-time excitation light. In this study, we developed a chemiluminescent probe named NE-CL for the detection of endogenous NE activity both in vitro and in vivo. The NE-CL consists of an acrylate-substituted dioxetane scaffold, a self-immolative linker, and a perfluorinated propionamide substrate that acts as the recognition group. In the presence of NE, a significant amount of chemiluminescence is generated by NE-CL with an impressive signal-to-noise ratio (∼342-fold) and high sensitivity (LOD∼20.8 ng/mL). Furthermore, the exceptional selectivity and appropriate luminescence duration time of NE-CL render it suitable for in vivo sensing. More importantly, NE-CL has been successfully utilized to differentiate various cell lines based on endogenous NE activity and visualize NE expression levels in mice inflammation model. These finding suggest that NE-CL possesses great potential for highly sensitive and accessible detection of NE-related diseases in the future.

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

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. 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. 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. We reviewed and included 132 articles conceptualizing neutrophil elastase activity and known inhibitors. 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.

Suppression of neutrophils by sodium exacerbates oxidative stress and arthritis.

Typical Western diet, rich in salt, contributes to autoimmune disease development. However, conflicting reports exist about the effect of salt on neutrophil effector functions, also in the context of arthritis. We investigated the effect of sodium chloride (NaCl) on neutrophil viability and functions in vitro, and in vivo employing the murine K/BxN-serum transfer arthritis (STA) model. The effects of NaCl and external reactive oxygen species (H2O2) were further examined on osteoclasts in vitro. Hypertonic sodium-rich media caused primary/secondary cell necrosis, altered the nuclear morphology, inhibited phagocytosis, degranulation, myeloperoxidase (MPO) peroxidation activity and neutrophil extracellular trap (NET) formation, while increasing total ROS production, mitochondrial ROS production, and neutrophil elastase (NE) activity. High salt diet (HSD) aggravated arthritis by increasing inflammation, bone erosion, and osteoclast differentiation, accompanied by increased NE expression and activity. Osteoclast differentiation was decreased with 25 mM NaCl or 100 nM H2O2 addition to isotonic media. In contrast to NaCl, external H2O2 had pro-resorptive effects in vitro. We postulate that in arthritis under HSD, increased bone erosion can be attributed to an enhanced oxidative milieu maintained by infiltrating neutrophils, rather than a direct effect of NaCl.

Lack of eosinophil extracellular trap formation due to failure of plasma membrane breakdown in the absence of elastase

AbstractActivated eosinophils are described to release eosinophil extracellular traps (EETs), which consist of the cell’s DNA covered with granule-derived antimicrobial peptides. Upon stimulation of eosinophils with the known EET-inducers phorbol 12-myristate 13-acetate, monosodium urate crystals, or Candida albicans, we observed that their plasma membrane became compromised, resulting in accessibility of the nuclear DNA for staining with the impermeable DNA dye Sytox Green. However, we did not observe any DNA decondensation or plasma membrane rupture by eosinophils, which sharply contrasts with neutrophil extracellular trap (NET) formation and the subsequent cell death known as NETosis. Neutrophil elastase (NE) activity is thought to be essential for the cleavage of histones and chromatin decondensation during NETosis. We observed that the neutrophils of a patient with a mutation in ELANE, leading to congenital neutropenia and NE deficiency, were unable to undergo NETosis. Taken together, we may suggest that the natural absence of any NE-like proteolytic activity in human eosinophils explains why EET formation is not observed, even when eosinophils become positive for an impermeable DNA dye in response to stimuli that induce NETosis in neutrophils.

Extracorporeal photopheresis induces NETosis in neutrophils derived from patients with chronic graft-vs-host disease.

Extracorporeal photopheresis (ECP) is considered an effective treatment for patients with chronic graft vs host disease (cGVHD) and demonstrates efficacy in ameliorating GVHD. The mechanism by which ECP acts against cGVHD is not fully understood. Preliminary observations have hinted at the potential involvement of neutrophil extracellular traps (NETs) formation in the pathogenesis of cGVHD. We aimed to assess the influence of ECP on the formation of NETs in patients with cGVHD as a potential mechanism in this setting. Patients treated with ECP for cGVHD at the Rabin Medical Center were included in this study. Blood samples were obtained at three different time points: before starting an ECP cycle, at the end of the first day of treatment, and 24 h following the initiation of the ECP treatment cycle. Neutrophils were harvested from all blood samples. NET formation was assessed by measurement of NET-bound specific neutrophil elastase activity and by immunofluorescence staining. Six patients (two females and four males) with cGVHD were included in the study. We observed a significant increase in NET formation among all six patients following ECP. Net-bound specific neutrophil elastase activity was elevated from a median value of 2.23 mU/mL (interquartile range [IQR] 2.06-2.47 mU/mL) at baseline to a median value of 13.06 mU/mL (IQR 10.27-15.97 mU/mL) immediately after the treatment and to a peak median value of 14.73 mU/mL (IQR 9.6-22.38 mU/mL) 24 h following the initiation of the ECP cycle. A qualitative assessment of NET formation using immunofluorescence staining has demonstrated markedly increased expression of citrullinated histone H3, a marker of NET formation, following ECP treatment. Our preliminary data indicate that ECP induces NET formation among patients with cGVHD. The contribution of increased NET formation to the therapeutic effect of cGVHD should be further investigated.

Longitudinal effects of elexacaftor/tezacaftor/ivacaftor on sputum viscoelastic properties, airway infection and inflammation in patients with cystic fibrosis.

Recent studies demonstrated that the triple combination cystic fibrosis transmembrane conductance regulator (CFTR) modulator therapy elexacaftor/tezacaftor/ivacaftor (ETI) improves lung function and reduces pulmonary exacerbations in cystic fibrosis (CF) patients with at least one F508del allele. However, effects of ETI on downstream consequences of CFTR dysfunction, i.e. abnormal viscoelastic properties of airway mucus, chronic airway infection and inflammation have not been studied. The aim of this study was to determine the longitudinal effects of ETI on airway mucus rheology, microbiome and inflammation in CF patients with one or two F508del alleles aged ≥12 years throughout the first 12 months of therapy. In this prospective observational study, we assessed sputum rheology, the microbiome, inflammation markers and proteome before and 1, 3 and 12 months after initiation of ETI. In total, 79 patients with CF and at least one F508del allele and 10 healthy controls were enrolled in this study. ETI improved the elastic modulus and viscous modulus of CF sputum at 3 and 12 months after initiation (all p<0.01). Furthermore, ETI decreased the relative abundance of Pseudomonas aeruginosa in CF sputum at 3 months and increased the microbiome α-diversity at all time points. In addition, ETI reduced interleukin-8 at 3 months (p<0.05) and free neutrophil elastase activity at all time points (all p<0.001), and shifted the CF sputum proteome towards healthy. Our data demonstrate that restoration of CFTR function by ETI improves sputum viscoelastic properties, chronic airway infection and inflammation in CF patients with at least one F508del allele over the first 12 months of therapy; however, levels close to healthy were not reached.

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.

Dipeptidyl peptidase-1 inhibition with brensocatib reduces the activity of all major neutrophil serine proteases in patients with bronchiectasis: results from the WILLOW trial

BackgroundBrensocatib is an oral, selective, reversible inhibitor of dipeptidyl peptidase-1 (DPP-1), responsible for activating neutrophil serine proteases (NSPs) including neutrophil elastase (NE), proteinase 3 (PR3), and cathepsin G (CatG). In chronic inflammatory lung diseases such as non-cystic fibrosis bronchiectasis (NCFBE), neutrophils accumulate in the airways resulting in excess active NSPs that cause damaging inflammation and lung destruction.MethodsThe 24-week WILLOW trial (NCT03218917) was a randomized, double-blind, placebo-controlled, parallel-group trial in patients with NCFBE conducted at 116 sites across 14 countries. In this trial, treatment with brensocatib was associated with improvements in clinical outcomes including time to first exacerbation, reduction in exacerbation frequency and a reduction in NE activity in sputum. An exploratory analysis of NE activity in white blood cell (WBC) extracts and NE, PR3 and CatG activity in sputum was conducted to further characterize brensocatib’s effect and identify potential correlated effects.ResultsNE, PR3 and CatG activities were reduced in sputum and NE activity was reduced in WBC extracts in a dose-dependent manner after four weeks of brensocatib treatment, with a return to baseline four weeks after the end of treatment. Brensocatib produced the greatest reduction in the sputum activity of CatG, followed by NE and then PR3. Positive correlations among the sputum NSPs were observed both at baseline and in response to treatment, with the strongest correlation among the sputum NSPs for NE and CatG.ConclusionsThese results suggest a broad anti-inflammatory effect of brensocatib underlying its clinical efficacy observed in NCFBE patients.Trial registration: The study was approved by the corresponding ethical review boards of all participating centers. The trial was approved by the Food and Drug Administration and registered at clinicaltrials.gov (NCT03218917) on July 17, 2017 and approved by the European Medicines Agency and registered at the European Union Clinical trials Register (EudraCT No. 2017-002533-32). An independent, external data and safety monitoring committee (comprising physicians with pulmonary expertise, a statistician experienced in the evaluation of clinical safety, and experts in periodontal disease and dermatology) reviewed all adverse events.

Neutrophil elastase decreases SARS-CoV-2 spike protein binding to human bronchial epithelia by clipping ACE-2 ectodomain from the epithelial surface

Patients with cystic fibrosis (CF) have decreased severity of SARS-CoV-2 infections, but the underlying cause is unknown. Patients with CF have high levels of neutrophil elastase (NE) in the airway. We examined whether respiratory epithelial angiotensin converting enzyme 2 (ACE-2), the receptor for the SARS-CoV-2 spike protein, is a proteolytic target of NE. Soluble ACE-2 (sACE-2) levels were quantified by ELISA in airway secretions and serum from patients with and without CF, and the association between sACE-2 levels and NE activity levels was evaluated in CF sputum. We determined that NE activity was directly correlated with increased ACE-2 in CF sputum. Additionally, primary human bronchial epithelial (HBE) cells, exposed to NE or control vehicle, were evaluated by western analysis for the release of cleaved ACE-2 ectodomain fragment into conditioned media, and by flow cytometry for the loss of cell surface ACE-2, its impact on SARS-CoV-2 spike protein binding. We found that NE treatment released ACE-2 ectodomain fragment from HBE and decreased spike protein binding to HBE. Furthermore, we performed NE treatment of recombinant ACE-2-Fc tagged protein in vitro to assess whether NE was sufficient to cleave recombinant ACE-2-Fc protein. Proteomic analysis identified, specific NE cleavage sites in the ACE-2 ectodomain that would result in loss of the putative N-terminal spike binding domain. Collectively, data support that NE plays a disruptive role in SARS-CoV-2 infection by catalyzing ACE-2 ectodomain shedding from the airway epithelia. This mechanism may reduce SARS-CoV-2 virus binding to respiratory epithelial cells and decrease severity of COVID19 infection.

Neutrophil metabolomics in severe COVID-19 reveal GAPDH as a suppressor of neutrophil extracellular trap formation

Severe COVID-19 is characterized by an increase in the number and changes in the function of innate immune cells including neutrophils. However, it is not known how the metabolome of immune cells changes in patients with COVID-19. To address these questions, we analyzed the metabolome of neutrophils from patients with severe or mild COVID-19 and healthy controls. We identified widespread dysregulation of neutrophil metabolism with disease progression including in amino acid, redox, and central carbon metabolism. Metabolic changes in neutrophils from patients with severe COVID-19 were consistent with reduced activity of the glycolytic enzyme GAPDH. Inhibition of GAPDH blocked glycolysis and promoted pentose phosphate pathway activity but blunted the neutrophil respiratory burst. Inhibition of GAPDH was sufficient to cause neutrophil extracellular trap (NET) formation which required neutrophil elastase activity. GAPDH inhibition increased neutrophil pH, and blocking this increase prevented cell death and NET formation. These findings indicate that neutrophils in severe COVID-19 have an aberrant metabolism which can contribute to their dysfunction. Our work also shows that NET formation, a pathogenic feature of many inflammatory diseases, is actively suppressed in neutrophils by a cell-intrinsic mechanism controlled by GAPDH.

Endobronchial optical coherence tomography or computed tomography for evaluating progression of bronchiectasis.

The early radiological signs of progression in bronchiectasis remain unclear. The objective of the present study was to compare endobronchial optical coherence tomography (EB-OCT) and chest computed tomography (CT) for the evaluation of radiological progression of bronchiectasis via stratification of the presence (TW+) or absence (TW-) of thickened-walled bronchioles surrounding dilated bronchi in patients with bronchiectasis based on CT, and determine the risk factors. In this prospective cohort study, we performed both chest CT and EB-OCT at baseline and 5-year follow-up, to compare changes in airway calibre metrics. We evaluated bacterial microbiology, sputum matrix metalloproteinase-9 levels and free neutrophil elastase activity at baseline. We compared clinical characteristics and airway calibre metrics between the TW+ and TW- groups. We ascertained radiological progression at 5 years via CT and EB-OCT. We recruited 75 patients between 2014 and 2017. At baseline, EB-OCT metrics (mean luminal diameter (p=0.017), inner airway area (p=0.005) and airway wall area (p=0.009) of seventh- to ninth-generation bronchioles) were significantly greater in the TW+ group than in the TW-group. Meanwhile, EB-OCT did not reveal bronchiole dilatation (compared with the same segment of normal bronchioles) surrounding nondilated bronchi on CT in the TW- group. At 5 years, 53.1% of patients in the TW+ group progressed to have bronchiectasis measured with EB-OCT, compared with only 3.3% in TW- group (p<0.05). 34 patients in the TW+ group demonstrated marked dilatation of medium-sized and small airways. Higher baseline neutrophil elastase activity and TW+ bronchioles on CT predicted progression of bronchiectasis. Thickened-walled bronchioles surrounding the dilated bronchi, identified with EB-OCT, indicates progression of bronchiectasis.

A Phase 2 randomised study to establish efficacy, safety and dosing of a novel oral cathepsin C inhibitor, BI 1291583, in adults with bronchiectasis: Airleaf.

New therapies are needed to prevent exacerbations, improve quality of life and slow disease progression in bronchiectasis. Inhibition of cathepsin C (CatC) activity has the potential to decrease activation of neutrophil-derived serine proteases in patients with bronchiectasis, thereby reducing airway inflammation, improving symptoms, reducing exacerbations and preventing further airway damage. Here we present the design of a phase 2 trial (Airleaf™; NCT05238675) assessing the efficacy and safety of a novel CatC inhibitor, BI 1291583, in adult patients with bronchiectasis. This multinational, randomised, double-blind, placebo-controlled, parallel-group, dose-finding study has a screening period of at least 6 weeks, a treatment period of 24-48 weeks and a follow-up period of 4 weeks. ∼240 adults with bronchiectasis of multiple aetiologies will be randomised to placebo once daily, or BI 1291583 1 mg once daily, 2.5 mg once daily or 5 mg once daily in a 2:1:1:2 ratio, stratified by Pseudomonas aeruginosa infection and maintenance use of macrolides. The primary efficacy objective is to evaluate the dose-response relationship for the three oral doses of BI 1291583 versus placebo on time to first pulmonary exacerbation up to Week 48 (the primary end-point). Efficacy will be assessed using exacerbations, patient-reported outcomes, measures of symptoms, sputum neutrophil elastase activity and pulmonary function testing. Safety assessment will include adverse event reporting, physical examination, monitoring of vital signs, safety laboratory parameters, 12-lead electrocardiogram, and periodontal and dermatological assessments. If efficacy and safety are demonstrated, results will support further investigation of BI 1291583 in phase 3 trials.

Diesel exhaust exposure impairs recovery of lung epithelial and cellular damage in murine model

Studies have investigated the relationship between diesel exhaust (DE) exposure and lung health, highlighting the potential for DE to induce pulmonary inflammation and oxidative stress. However, the resolution of inflammation upon withdrawal of DE exposure needs further investigation. Therefore, resolution of diesel exhaust-induced lung damage was studied in the murine model. Mice (6 weeks) were divided into three groups. Group 1 (control) mice were exposed to filtered air, Group 2 (DE) mice were exposed to DE (5.1 ± 0.7 mg/m3) & Group 3 (DE-FA) mice were exposed to DE followed by filtered air exposure. Airway hyper-responsiveness was recorded after 24 h of the last exposure. BALF and lung samples were collected for cytokine estimation, immunobiological assays, and western blot analysis. DE exposure showed an increase in lung resistance thereby causing alteration in lung function parameters (p < 0.05) which was restored in the DE-FA group. BALF analysis showed a significant increase in total cell count and protein content in DE with no resolution in DE-FA groups (p < 0.05). Lung histology showed no reduction in the bronchiolar thickness and damage in the DE-FA group suggesting irreversible lung damage (p < 0.05). The significant increase in inflammatory cytokine levels, and collagen deposition showed persistent inflammatory phase and lung damage in the DE-FA group(p < 0.05). ZO-1 was significantly decreased in both test groups indicating disintegrated lung epithelium where in claudin-5 expression showed increased lung permeability. A significant increase in neutrophil elastase activity and decreased expression of, Elafin, resulted in lung epithelial damage in the DE-FA group. Lung injury marker alpha1-antitrypsin was increased in DE-FA groups indicating an immune defense mechanism against neutrophil elastase. The study showed that DE exposure causes persistent lung damage via neutrophil elastase-associated disruption of the epithelial barrier integrity and membrane dysfunction.

Heparin, Low Molecular Weight Heparin, and Non-Anticoagulant Derivatives for the Treatment of Inflammatory Lung Disease.

Unfractionated heparin has multiple pharmacological activities beyond anticoagulation. These anti-inflammatory, anti-microbial, and mucoactive activities are shared in part by low molecular weight and non-anticoagulant heparin derivatives. Anti-inflammatory activities include inhibition of chemokine activity and cytokine synthesis, inhibitory effects on the mechanisms of adhesion and diapedesis involved in neutrophil recruitment, inhibition of heparanase activity, inhibition of the proteases of the coagulation and complement cascades, inhibition of neutrophil elastase activity, neutralisation of toxic basic histones, and inhibition of HMGB1 activity. This review considers the potential for heparin and its derivatives to treat inflammatory lung disease, including COVID-19, ALI, ARDS, cystic fibrosis, asthma, and COPD via the inhaled route.

The Direct Anti-Virulence but Not Bactericidal Activity of Human Neutrophil Elastase against Moraxella catarrhalis.

Neutrophil elastase (NE) contributes to innate antibacterial defense at both the intracellular (phagocytosis) and extracellular (degranulation, NETosis) levels. Moraxella catarrhalis, a human respiratory pathogen, can exist in an inflammatory milieu which contains NE. No data are available on the action of NE against M. catarrhalis or on the counteraction of NE-dependent host defenses by this pathogen. Using time-kill assays we found that bacteria are able to survive and replicate in the presence of NE. Transmission electron microscopy and flow cytometry studies with NE-treated bacteria revealed that while NE admittedly destabilizes the outer membrane leaflet, it does not cause cytoplasmic membrane rupture, suggesting that the enzyme does not target components that are essential for cell integrity. Using LC-MS/MS spectroscopy we determined that NE cleaved at least three virulent surface proteins in outer membrane vesicles (OMVs) of M. catarrhalis, including OMP CD, McaP, and TbpA. The cleavage of OMP CD contributes to the significant decrease in resistance to serum complement in the complement-resistant strain Mc6. The cleavage of McaP did not cause any sensitization to erythromycin nor did NE disturb its drug action. Identifying NE as a novel but subtle anti-virulence agent together with its extracellularly not-efficient bactericidal activity against M. catarrhalis may facilitate the pathogen's existence in the airways under inflammation.

Selective and sensitive measurement of human neutrophil elastase in clinical samples based on a novel assay principle for protease activity measurement

Imbalances between proteases and protease inhibitors have been associated with several pathological conditions including emphysema as seen in α1-antitrypsin deficiency. For this pathological condition, unimpeded neutrophil elastase activity has been ascribed a pivotal role in the destruction of lung tissue and thus in disease progression. Therefore, low, or non-quantifiable neutrophil elastase (NE) activity levels determined in bronchoalveolar lavage solutions indicate the success of α1-antitrypsin (AAT) augmentation therapy as NE activity will be erased. To overcome the known limitations of available elastase activity assays regarding sensitivity and selectivity, we developed a new elastase activity assay, which fundamentally relies on the highly specific complex formation between AAT and active elastase. Plate-bound AAT captured active elastase from the sample undergoing complex formation, followed by the immunological detection of human NE. This assay principle facilitated the measurement of low pM amounts of active human NE. The data of the assay performance check demonstrated adequate accuracy and precision profiles meeting currently accepted best practices for this activity assay, which can be classified as a ligand-binding assay. Furthermore, spike-recovery studies at low human NE levels, carried out for three human bronchoalveolar samples, resulted in recoveries within the 100 ± 20% range, while good linearity and parallelism of the samples’ dilution-response curves was observed. Altogether, complemented by the data of selectivity and robustness studies and the accuracy and precision profile obtained in buffer, this newly developed human NE activity assay was demonstrated to perform accurately and precisely in clinically relevant samples.

Benefit-risk assessment of brensocatib for treatment of non-cystic fibrosis bronchiectasis.

Brensocatib is a novel anti-inflammatory therapy in development for bronchiectasis treatment. Phase 2 WILLOW trial data demonstrate a low number needed to treat and negative number needed to harm, suggesting a favourable benefit-risk profile. https://bit.ly/3SbisW3.

2-deoxy-2-[18F]fluoro-D-glucose Positron Emission Tomography to Monitor Lung Inflammation and Therapeutic Response to Dexamethasone in a Murine Model of Acute Lung Injury.

To image inflammation and monitor therapeutic response to anti-inflammatory intervention using 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) positron emission tomography (PET) in a preclinical model of acute lung injury (ALI). Mice were intratracheally administered lipopolysaccharide (LPS, 2.5mg/kg) to induce ALI or phosphate-buffered saline as the vehicle control. A subset of mice in the ALI group received two intraperitoneal doses of dexamethasone 1 and 24h after LPS. [18F]FDG PET/CT was performed 2days after the induction of ALI. [18F]FDG uptake in the lungs was quantified by PET (%ID/mLmean and standardized uptake value (SUVmean)) and ex vivo γ-counting (%ID/g). The severity of lung inflammation was determined by quantifying the protein level of inflammatory cytokines/chemokines and the activity of neutrophil elastase and glycolytic enzymes. In separate groups of mice, flow cytometry was performed to estimate the contribution of individual immune cell types to the total pulmonary inflammatory cell burden under different treatment conditions. Lung uptake of [18F]FDG was significantly increased during LPS-induced ALI, and a decreased [18F]FDG uptake was observed following dexamethasone treatment to an intermediate level between that of LPS-treated and control mice. Protein expression of inflammatory biomarkers and the activity of neutrophil elastase and glycolytic enzymes were increased in the lungs of LPS-treated mice versus those of control mice, and correlated with [18F]FDG uptake. Furthermore, dexamethasone-induced decreases in cytokine/chemokine protein levels and enzyme activities correlated with [18F]FDG uptake. Neutrophils were the most abundant cells in LPS-induced ALI, and the pattern of total cell burden during ALI with or without dexamethasone therapy mirrored that of [18F]FDG uptake. [18F]FDG PET noninvasively detects lung inflammation in ALI and its response to anti-inflammatory therapy in a preclinical model. However, high [18F]FDG uptake by bone, brown fat, and myocardium remains a technical limitation for quantification of [18F]FDG in the lungs.

Necrostatin-1 Alleviates Diffuse Pulmonary Haemorrhage by Preventing the Release of NETs via Inhibiting NE/GSDMD Activation in Murine Lupus.

Diffuse alveolar haemorrhage (DAH) is a rapidly developing condition owing to a lack of effective treatment and resulting in a high mortality rate in systemic lupus erythematosus (SLE). Neutrophil extracellular traps (NETs) contain numerous antigens and proinflammatory substances that directly damage the vascular endothelium and aggravate vascular inflammation, which is considered an important pathogenic factor of DAH in SLE. Therefore, blocking the release of NETs from neutrophils is an important target for the treatment of DAH in SLE. In this study, we investigated whether the inhibition of neutrophils releasing NETs could relieve DAH in SLE. Necrostatin-1 (Nec-1), a small molecule, has been reported to inhibit the release of NETs by neutrophils. In vitro experiments revealed that Nec-1 inhibited alveolar epithelial cell damage by preventing the release of NETs. Furthermore, vivo studies showed that Nec-1 alleviated lupus pulmonary haemorrhage in mice by reducing lung pathology severity, body weight, and serum inflammatory cytokine levels. Mechanistically, Nec-1 prevented NET release by inhibiting neutrophil elastase (NE) activation and N-Gasdermin D (N-GSDMD) expression. Additionally, immunohistochemistry and immunofluorescence findings showed that Nec-1 decreased NE expression in the lung tissues of mice with lupus pulmonary haemorrhage. Thus, NETs released by neutrophils contributed to the pathogenesis of DAH in SLE, and Nec-1 showed protective effects by the inhibition of NET production via the reduction of NE activation and N-GSDMD expression.