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Molecules (Basel, Switzerland) May 2023During a search for natural inflammatory inhibitors, 1--acetylbritannilactone (ABL), a sesquiterpene lactone, was isolated from the flowers of . ABL significantly...
During a search for natural inflammatory inhibitors, 1--acetylbritannilactone (ABL), a sesquiterpene lactone, was isolated from the flowers of . ABL significantly inhibited human neutrophil elastase (HNE) with a half-maximal inhibitory concentration (IC) of 3.2 ± 0.3 µM, thus did so more effectively than the positive control material (epigallocatechin gallate) (IC 7.2 ± 0.5 µM). An enzyme kinetic study was performed. ABL noncompetitively inhibited HNE with an inhibition constant of 2.4 µM. ABL inhibited lipopolysaccharide-induced nitric oxide and prostaglandin E production by RAW 264.7 cells in a dose-dependent manner, as well as the protein-level expression of inducible nitric oxide synthase and cyclooxygenase-2. The anti-inflammatory effect of ABL was confirmed using a transgenic Tg(:EGFP) zebrafish larval model. The exposure of the larvae to ABL inhibited neutrophil recruitment to the site of injury after tail fin amputation.
Topics: Animals; Mice; Humans; Inula; Zebrafish; RAW 264.7 Cells; Leukocyte Elastase; Inflammation; Lactones; Flowers
PubMed: 37298794
DOI: 10.3390/molecules28114320 -
The Journal of Biological Chemistry Aug 2023Human neutrophil elastase (HNE) plays a pivotal role in innate immunity, inflammation, and tissue remodeling. Aberrant proteolytic activity of HNE contributes to organ...
Human neutrophil elastase (HNE) plays a pivotal role in innate immunity, inflammation, and tissue remodeling. Aberrant proteolytic activity of HNE contributes to organ destruction in various chronic inflammatory diseases including emphysema, asthma, and cystic fibrosis. Therefore, elastase inhibitors could alleviate the progression of these disorders. Here, we used the systematic evolution of ligands by exponential enrichment to develop ssDNA aptamers that specifically target HNE. We determined the specificity of the designed inhibitors and their inhibitory efficacy against HNE using biochemical and in vitro methods, including an assay of neutrophil activity. Our aptamers inhibit the elastinolytic activity of HNE with nanomolar potency and are highly specific for HNE and do not target other tested human proteases. As such, this study provides lead compounds suitable for the evaluation of their tissue-protective potential in animal models.
Topics: Humans; Cystic Fibrosis; Emphysema; Leukocyte Elastase; Neutrophils; Serine Proteinase Inhibitors; Aptamers, Nucleotide; Sensitivity and Specificity; Enzyme Activation; Proteolysis; Cells, Cultured
PubMed: 37286041
DOI: 10.1016/j.jbc.2023.104889 -
Biomaterials Advances Aug 2023In chronic wound (CW) scenarios, Staphylococcus aureus-induced infections are very prevalent. This leads to abnormal inflammatory processes, in which proteolytic...
Sodium alginate/polycaprolactone co-axial wet-spun microfibers modified with N-carboxymethyl chitosan and the peptide AAPV for Staphylococcus aureus and human neutrophil elastase inhibition in potential chronic wound scenarios.
In chronic wound (CW) scenarios, Staphylococcus aureus-induced infections are very prevalent. This leads to abnormal inflammatory processes, in which proteolytic enzymes, such as human neutrophil elastase (HNE), become highly expressed. Alanine-Alanine-Proline-Valine (AAPV) is an antimicrobial tetrapeptide capable of suppressing the HNE activity, restoring its expression to standard rates. Here, we proposed the incorporation of the peptide AAPV within an innovative co-axial drug delivery system, in which the peptide liberation was controlled by N-carboxymethyl chitosan (NCMC) solubilization, a pH-sensitive antimicrobial polymer effective against Staphylococcus aureus. The microfibers' core was composed of polycaprolactone (PCL), a mechanically resilient polymer, and AAPV, while the shell was made of the highly hydrated and absorbent sodium alginate (SA) and NCMC, responsive to neutral-basic pH (characteristic of CW). NCMC was loaded at twice its minimum bactericidal concentration (6.144 mg/mL) against S. aureus, while AAPV was loaded at its maximum inhibitory concentration against HNE (50 μg/mL), and the production of fibers with a core-shell structure, in which all components could be detected (directly or indirectly), was confirmed. Core-shell fibers were characterized as flexible and mechanically resilient, and structurally stable after 28-days of immersion in physiological-like environments. Time-kill kinetics evaluations revealed the effective action of NCMC against S. aureus, while elastase inhibitory activity examinations proved the ability of AAPV to reduce HNE levels. Cell biology testing confirmed the safety of the engineered fiber system for human tissue contact, with fibroblast-like cells and human keratinocytes maintaining their morphology while in contact with the produced fibers. Data confirmed the engineered drug delivery platform as potentially effective for applications in CW care.
Topics: Humans; Alginates; Chitosan; Leukocyte Elastase; Peptides; Polymers; Staphylococcal Infections; Staphylococcus aureus; Valine; Wounds and Injuries; Wound Healing
PubMed: 37285725
DOI: 10.1016/j.bioadv.2023.213488 -
The Journal of Biological Chemistry Jul 2023Extracellular adherence protein domain (EAP) proteins are high-affinity, selective inhibitors of neutrophil serine proteases (NSP), including cathepsin-G (CG) and...
Extracellular adherence protein domain (EAP) proteins are high-affinity, selective inhibitors of neutrophil serine proteases (NSP), including cathepsin-G (CG) and neutrophil elastase (NE). Most Staphylococcus aureus isolates encode for two EAPs, EapH1 and EapH2, that contain a single functional domain and share 43% identity with one another. Although structure/function investigations from our group have shown that EapH1 uses a globally similar binding mode to inhibit CG and NE, NSP inhibition by EapH2 is incompletely understood due to a lack of NSP/EapH2 cocrystal structures. To address this limitation, we further studied NSP inhibition by EapH2 in comparison with EapH1. Like its effects on NE, we found that EapH2 is a reversible, time-dependent, and low nanomolar affinity inhibitor of CG. We characterized an EapH2 mutant which suggested that the CG binding mode of EapH2 is comparable to EapH1. To test this directly, we used NMR chemical shift perturbation to study EapH1 and EapH2 binding to CG and NE in solution. Although we found that overlapping regions of EapH1 and EapH2 were involved in CG binding, we found that altogether distinct regions of EapH1 and EapH2 experienced changes upon binding to NE. An important implication of this observation is that EapH2 might be capable of binding and inhibiting CG and NE simultaneously. We confirmed this unexpected feature by solving crystal structures of the CG/EapH2/NE complex and demonstrating their functional relevance through enzyme inhibition assays. Together, our work defines a new mechanism of simultaneous inhibition of two serine proteases by a single EAP protein.
Topics: Bacterial Proteins; Cathepsin G; Immune Evasion; Leukocyte Elastase; Neutrophils; Serine Proteases; Staphylococcus aureus
PubMed: 37269950
DOI: 10.1016/j.jbc.2023.104878 -
Respiratory Research May 2023Brensocatib is an oral, selective, reversible inhibitor of dipeptidyl peptidase-1 (DPP-1), responsible for activating neutrophil serine proteases (NSPs) including... (Randomized Controlled Trial)
Randomized Controlled Trial
Dipeptidyl peptidase-1 inhibition with brensocatib reduces the activity of all major neutrophil serine proteases in patients with bronchiectasis: results from the WILLOW trial.
BACKGROUND
Brensocatib 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.
METHODS
The 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.
RESULTS
NE, 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.
CONCLUSIONS
These 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.
Topics: Humans; Serine Proteases; Neutrophils; Salix; Bronchiectasis; Leukocyte Elastase; Cystic Fibrosis; Myeloblastin; Dipeptidyl-Peptidases and Tripeptidyl-Peptidases
PubMed: 37198686
DOI: 10.1186/s12931-023-02444-z -
Diagnostics (Basel, Switzerland) Apr 2023Sepsis, the leading cause of mortality in hospitals, currently lacks effective early diagnostics. A new cellular host response test, the IntelliSep test, may provide an...
Sepsis, the leading cause of mortality in hospitals, currently lacks effective early diagnostics. A new cellular host response test, the IntelliSep test, may provide an indicator of the immune dysregulation characterizing sepsis. The objective of this study was to examine the correlation between the measurements performed using this test and biological markers and processes associated with sepsis. Phorbol myristate acetate (PMA), an agonist of neutrophils known to induce neutrophil extracellular trap (NET) formation, was added to whole blood of healthy volunteers at concentrations of 0, 200, and 400 nM and then evaluated using the IntelliSep test. Separately, plasma from a cohort of subjects was segregated into Control and Diseased populations and tested for levels of NET components (citrullinated histone (cit-H3) DNA and neutrophil elastase (NE) DNA) using customized ELISA assays and correlated with ISI scores from the same patient samples. Significant increases in IntelliSep Index (ISI) scores were observed with increasing concentrations of PMA in healthy blood (0 and 200: < 10; 0 and 400: < 10). Linear correlation was observed between the ISI and quantities of NE DNA and Cit-H3 DNA in patient samples. Together these experiments demonstrate that the IntelliSep test is associated with the biological processes of leukocyte activation and NETosis and may indicate changes consistent with sepsis.
PubMed: 37189536
DOI: 10.3390/diagnostics13081435 -
The Journal of Biological Chemistry Jun 2023Patients with cystic fibrosis (CF) have decreased severity of severe acute respiratory syndrome-like coronavirus-2 (SARS-CoV-2) infections, but the underlying cause is...
Patients with cystic fibrosis (CF) have decreased severity of severe acute respiratory syndrome-like coronavirus-2 (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 levels were quantified by ELISA in airway secretions and serum from patients with and without CF, the association between soluble ACE-2 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, 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 the severity of COVID19 infection.
Topics: Humans; Angiotensin-Converting Enzyme 2; COVID-19; Cystic Fibrosis; Leukocyte Elastase; Protein Binding; Proteomics; Respiratory Mucosa; SARS-CoV-2; Spike Glycoprotein, Coronavirus
PubMed: 37187291
DOI: 10.1016/j.jbc.2023.104820 -
International Journal of Molecular... Apr 2023Intense neutrophil infiltration into the liver is a characteristic of acetaminophen-induced acute liver injury. Neutrophil elastase is released by neutrophils during...
Intense neutrophil infiltration into the liver is a characteristic of acetaminophen-induced acute liver injury. Neutrophil elastase is released by neutrophils during inflammation. To elucidate the involvement of neutrophil elastase in acetaminophen-induced liver injury, we investigated the efficacy of a potent and specific neutrophil elastase inhibitor, sivelestat, in mice with acetaminophen-induced acute liver injury. Intraperitoneal administration of 750 mg/kg of acetaminophen caused severe liver damage, such as elevated serum transaminase levels, centrilobular hepatic necrosis, and neutrophil infiltration, with approximately 50% mortality in BALB/c mice within 48 h of administration. However, in mice treated with sivelestat 30 min after the acetaminophen challenge, all mice survived, with reduced serum transaminase elevation and diminished hepatic necrosis. In addition, mice treated with sivelestat had reduced NOS-II expression and hepatic neutrophil infiltration after the acetaminophen challenge. Furthermore, treatment with sivelestat at 3 h after the acetaminophen challenge significantly improved survival. These findings indicate a new clinical application for sivelestat in the treatment of acetaminophen-induced liver failure through mechanisms involving the regulation of neutrophil migration and NO production.
Topics: Mice; Animals; Acetaminophen; Leukocyte Elastase; Mice, Inbred BALB C; Liver Diseases; Transaminases; Chemical and Drug Induced Liver Injury; Necrosis
PubMed: 37175553
DOI: 10.3390/ijms24097845 -
JVS-vascular Science 2023Metformin treatment attenuates experimental abdominal aortic aneurysm (AAA) formation, as well as reduces clinical AAA diameter enlargement in patients with diabetes....
OBJECTIVE
Metformin treatment attenuates experimental abdominal aortic aneurysm (AAA) formation, as well as reduces clinical AAA diameter enlargement in patients with diabetes. The mechanisms of metformin-mediated aneurysm suppression, and its efficacy in suppressing established experimental aneurysms, remain uncertain.
METHODS
Experimental AAAs were created in male C57BL/6J mice via intra-aortic infusion of porcine pancreatic elastase. Metformin alone (250 mg/kg), or metformin combined with the 5' AMP-activated protein kinase (AMPK) antagonist Compound C (10 mg/kg), were administered to respective mouse cohorts daily beginning 4 days following AAA induction. Further AAA cohorts received either the AMPK agonist AICA riboside (500 mg/kg) as positive, or vehicle (saline) as negative, controls. AAA progression in all groups was assessed via serial in vivo ultrasonography and histopathology at sacrifice. Cytokine-producing T cells and myeloid cellularity were determined by flow cytometric analyses.
RESULTS
Metformin limited established experimental AAA progression at 3 (-85%) and 10 (-68%) days following treatment initiation compared with saline control. Concurrent Compound C treatment reduced this effect by approximately 50%. In metformin-treated mice, reduced AAA progression was associated with relative elastin preservation, smooth muscle cell preservation, and reduced mural leukocyte infiltration and neoangiogenesis compared with vehicle control group. Metformin also resulted in reduced interferon-γ-, but not interleukin-10 or -17, producing splenic T cells in aneurysmal mice. Additionally, metformin therapy increased circulating and splenic inflammatory monocytes (CD11bLy-6C), but not neutrophils (CD11bLy-6G), with no effect on respective bone marrow cell populations.
CONCLUSIONS
Metformin treatment suppresses existing experimental AAA progression in part via AMPK agonist activity, limiting interferon-γ-producing T cell differentiation while enhancing circulating and splenic inflammatory monocyte retention.
PubMed: 37168662
DOI: 10.1016/j.jvssci.2023.100102 -
The Journal of Biological Chemistry Jun 2023Pneumococcus is the main cause of bacterial pneumonia. Pneumococcal infection has been shown to cause elastase, an intracellular host defense factor, to leak from...
Pneumococcus is the main cause of bacterial pneumonia. Pneumococcal infection has been shown to cause elastase, an intracellular host defense factor, to leak from neutrophils. However, when neutrophil elastase (NE) leaks extracellularly, it can degrade host cell surface proteins such as epidermal growth factor receptor (EGFR) and potentially disrupt the alveolar epithelial barrier. In this study, we hypothesized that NE degrades the extracellular domain (ECD) of EGFR in alveolar epithelial cells and inhibits alveolar epithelial repair. Using SDS-PAGE, we showed that NE degraded the recombinant EGFR ECD and its ligand epidermal growth factor, and that the degradation of these proteins was counteracted by NE inhibitors. Furthermore, we confirmed the degradation by NE of EGFR expressed in alveolar epithelial cells in vitro. We showed that intracellular uptake of epidermal growth factor and EGFR signaling was downregulated in alveolar epithelial cells exposed to NE and found that cell proliferation was inhibited in these cells These negative effects of NE on cell proliferation were abolished by NE inhibitors. Finally, we confirmed the degradation of EGFR by NE in vivo. Fragments of EGFR ECD were detected in bronchoalveolar lavage fluid from pneumococcal pneumonia mice, and the percentage of cells positive for a cell proliferation marker Ki67 in lung tissue was reduced. In contrast, administration of an NE inhibitor decreased EGFR fragments in bronchoalveolar lavage fluid and increased the percentage of Ki67-positive cells. These findings suggest that degradation of EGFR by NE could inhibit the repair of alveolar epithelium and cause severe pneumonia.
Topics: Animals; Mice; Bronchoalveolar Lavage Fluid; Epithelial Cells; ErbB Receptors; Ki-67 Antigen; Leukocyte Elastase; Lung; Pneumonia, Pneumococcal; Proteinase Inhibitory Proteins, Secretory
PubMed: 37119853
DOI: 10.1016/j.jbc.2023.104760