Journal: Frontiers in Immunology

Article Title: Neutrophil Elastase Subverts the Immune Response by Cleaving Toll-Like Receptors and Cytokines in Pneumococcal Pneumonia

doi: 10.3389/fimmu.2018.00732

Figure Lengend Snippet: Neutrophil elastase (NE) downregulates cytokine gene transcription in macrophages stimulated with TLR agonists. THP-1-derived macrophages were stimulated with HK-Spn D39 or Escherichia coli LPS (100 ng/mL) in the presence or absence of hNE (250–500 mU/mL) and/or SSH (100 µg/mL) for 4 h under serum-free conditions. Real-time PCR was performed to quantify TNF, IL6 , and IL8 mRNA in the macrophages exposed to these stimuli. The relative quantity of these cytokine mRNAs was normalized to the relative quantity of GAPDH mRNA. Data represent the mean ± SD of quadruplicate experiments and were evaluated using one-way analysis of variance with Tukey’s multiple-comparisons test. *Significantly different within the same activation status at P < 0.05. HK-Spn, heat-killed S. pneumoniae ; LPS, lipopolysaccharide; hNE, human neutrophil elastase; SSH, sivelestat sodium hydrate; TLR, toll-like receptor; TNF, tumor necrosis factor.

Article Snippet: After 48 h incubation, the cells were washed with RPMI 1640 and cultured further in RPMI 1640 for 12 h. Then, cells were stimulated with heat-killed S. pneumoniae (HK-Spn) or Escherichia coli lipopolysaccharide (LPS) (100 ng/mL; Sigma-Aldrich, St. Louis, MO, USA) in the presence or absence of human neutrophil elastase (hNE; Innovative Research, Novi, MI, USA) and the NE inhibitor sivelestat (100 μg/mL; ONO Pharmaceutical Co., Osaka, Japan) for 3 h under serum-free conditions.

Techniques: Derivative Assay, Real-time Polymerase Chain Reaction, Activation Assay

Journal: Frontiers in Immunology

Article Title: Neutrophil Elastase Subverts the Immune Response by Cleaving Toll-Like Receptors and Cytokines in Pneumococcal Pneumonia

doi: 10.3389/fimmu.2018.00732

Figure Lengend Snippet: Neutrophil elastase (NE) cleaves TLRs and MD2 on macrophages. (A,B) THP-1-derived macrophages were cultured in serum free RPMI 1640 and exposed to hNE (125–500 mU/mL) for 4 h. (A) Representative fluorescence microscopy images of untreated and hNE-treated (500 mU/mL) macrophages stained for DNA (DAPI; blue) and TLRs (green). (B) TLR2, TLR4, and TLR-related molecules were determined by western blot analysis. (C) Recombinant human (rh) TLR2, rhTLR4, and rhMD2 were exposed to hNE (125–500 mU/mL) in the presence or absence of SSH (100 µg/mL) for 3 h and determined by western blot analysis. MD2, myeloid differentiation factor 2; hNE, human neutrophil elastase; SSH, sivelestat sodium hydrate; TLR, toll-like receptor.

Article Snippet: After 48 h incubation, the cells were washed with RPMI 1640 and cultured further in RPMI 1640 for 12 h. Then, cells were stimulated with heat-killed S. pneumoniae (HK-Spn) or Escherichia coli lipopolysaccharide (LPS) (100 ng/mL; Sigma-Aldrich, St. Louis, MO, USA) in the presence or absence of human neutrophil elastase (hNE; Innovative Research, Novi, MI, USA) and the NE inhibitor sivelestat (100 μg/mL; ONO Pharmaceutical Co., Osaka, Japan) for 3 h under serum-free conditions.

Techniques: Derivative Assay, Cell Culture, Fluorescence, Microscopy, Staining, Western Blot, Recombinant

Journal: Frontiers in Immunology

Article Title: Neutrophil Elastase Subverts the Immune Response by Cleaving Toll-Like Receptors and Cytokines in Pneumococcal Pneumonia

doi: 10.3389/fimmu.2018.00732

Figure Lengend Snippet: Neutrophil elastase (NE) degrades TNF, IL-6, and IL-8 (A) . THP-1-derived macrophages were exposed to hNE (125–500 mU/mL) in the presence or absence of HK-Spn or LPS (100 ng/mL) for 4 h. TNF, IL-6, and IL-8 concentrations in the culture supernatants were determined by ELISA. Data represent the mean ± SD of quadruplicate experiments and were evaluated using one-way analysis of variance with Tukey’s multiple-comparisons test. *Significantly different within the same activation status at P < 0.05. (B) rhTNF, rhIL-6, and rhIL-8 were exposed to hNE (125–500 mU/mL) in the presence or absence of SSH (100 µg/mL) for 3 h and determined by western blot analysis. ELISA, enzyme-linked immunosorbent assay; HK-Spn, heat-killed S. pneumoniae ; LPS, lipopolysaccharide; hNE, human neutrophil elastase; rh, recombinant human; SSH, sivelestat sodium hydrate; IL, interleukin; TNF, tumor necrosis factor.

Article Snippet: After 48 h incubation, the cells were washed with RPMI 1640 and cultured further in RPMI 1640 for 12 h. Then, cells were stimulated with heat-killed S. pneumoniae (HK-Spn) or Escherichia coli lipopolysaccharide (LPS) (100 ng/mL; Sigma-Aldrich, St. Louis, MO, USA) in the presence or absence of human neutrophil elastase (hNE; Innovative Research, Novi, MI, USA) and the NE inhibitor sivelestat (100 μg/mL; ONO Pharmaceutical Co., Osaka, Japan) for 3 h under serum-free conditions.

Techniques: Derivative Assay, Enzyme-linked Immunosorbent Assay, Activation Assay, Western Blot, Recombinant

Journal: Frontiers in Immunology

Article Title: Neutrophil Elastase Subverts the Immune Response by Cleaving Toll-Like Receptors and Cytokines in Pneumococcal Pneumonia

doi: 10.3389/fimmu.2018.00732

Figure Lengend Snippet: Multiplex assay reveals that neutrophil elastase (NE) degrades various cytokines. Human cytokine/chemokine cocktail was exposed to hNE (125–500 mU/mL) in the presence or absence of SSH (100 µg/mL) for 3 h. Then, cytokines were detected using a multiplex assay. Data represent the mean ± SD of quadruplicate experiments and were evaluated using two-way ANOVA. *Significantly different from the hNE-untreated control at P < 0.05. † Significantly different from the hNE-treated (500 mU/mL) group in the absence of SSH at P < 0.05. hNE, human neutrophil elastase; SSH, sivelestat sodium hydrate.

Article Snippet: After 48 h incubation, the cells were washed with RPMI 1640 and cultured further in RPMI 1640 for 12 h. Then, cells were stimulated with heat-killed S. pneumoniae (HK-Spn) or Escherichia coli lipopolysaccharide (LPS) (100 ng/mL; Sigma-Aldrich, St. Louis, MO, USA) in the presence or absence of human neutrophil elastase (hNE; Innovative Research, Novi, MI, USA) and the NE inhibitor sivelestat (100 μg/mL; ONO Pharmaceutical Co., Osaka, Japan) for 3 h under serum-free conditions.

Techniques: Multiplex Assay, Control

Journal: Frontiers in Immunology

Article Title: Neutrophil Elastase Subverts the Immune Response by Cleaving Toll-Like Receptors and Cytokines in Pneumococcal Pneumonia

doi: 10.3389/fimmu.2018.00732

Figure Lengend Snippet: Administration of NE inhibitor increases cytokine level and decreases bacterial load in BALF. BALB/c mice (seven mice each) were intratracheally infected with Streptococcus pneumoniae D39 (2 × 10 8 CFU in 50 µL PBS). Unchallenged naive mice (Sham group) were administered PBS only. NE inhibitor (SSH group; 50 mg/kg) or PBS (PBS group) was administrated intraperitoneally to the infected mice every 6 h. (A–C) Groups of animals were sacrificed at 18 h postinfection. (A) BALF samples were plated onto blood-agar plates and cultured aerobically for enumerating recovered CFU. Data represent the mean ± SD and were evaluated using unpaired t tests. *Significantly different from the infected control group at P < 0.05. (B) The levels of TNF and IL-6 in BALF were determined by using ELISA kits. (C) Serum TNF and IL-6 levels were determined. (B,C) Data represent the mean ± SD and were evaluated using one-way analysis of variance with Tukey’s multiple-comparisons test. *Significantly different from the infected control group at P < 0.05. (D) Survival of BALB/c (twenty mice each) mice was monitored following the intratracheal infection with S. pneumoniae (5 × 10 8 CFU) in the presence or absence of intraperitoneal administration of an NE inhibitor. Statistical analysis was performed with log-rank test. *Significantly different from the SSH-untreated control at P < 0.001. BALF, bronchoalveolar lavage fluid; CFUs, colony forming units; ELISA, enzyme-linked immunosorbent assay; NE, neutrophil elastase; PBS, phosphate buffered saline; SSH, sivelestat sodium hydrate; IL, interleukin; TNF, tumor necrosis factor.

Article Snippet: After 48 h incubation, the cells were washed with RPMI 1640 and cultured further in RPMI 1640 for 12 h. Then, cells were stimulated with heat-killed S. pneumoniae (HK-Spn) or Escherichia coli lipopolysaccharide (LPS) (100 ng/mL; Sigma-Aldrich, St. Louis, MO, USA) in the presence or absence of human neutrophil elastase (hNE; Innovative Research, Novi, MI, USA) and the NE inhibitor sivelestat (100 μg/mL; ONO Pharmaceutical Co., Osaka, Japan) for 3 h under serum-free conditions.

Techniques: Infection, Cell Culture, Control, Enzyme-linked Immunosorbent Assay, Saline

Journal: The Journal of Biological Chemistry

Article Title: Neutrophil Elastase Acts as a Biased Agonist for Proteinase-activated Receptor-2 (PAR 2 ) *

doi: 10.1074/jbc.M110.201988

Figure Lengend Snippet: Elafin inhibition of NE abrogates activation of PAR2-dependent p44/42 MAPK activation. PAR2-expressing KNRK cells were serum-starved and stimulated with NE that had been preincubated with different concentrations of elafin for 10 min. A, representative image showing activation of p44/42 MAPK by NE in PAR2-KNRK cells and the attenuation of NE-stimulated MAPK signal by the NE inhibitor elafin. B, histogram showing densitometry analysis of multiple scanned Western blot images to quantify the percentage increase over base line of PAR2-KNRK p44/42 activation by NE in the presence or absence of elafin. Increases in p44/42 MAPK phosphorylation (p-p44/42) were normalized to total p44/42 MAPK (t-p44/42) signal in the same samples and expressed as a percentage increase over base-line values (mean ± S.E. (error bars), n = 3; *, significant decrease in signal compared with the NE (3 units/ml) samples; p < 0.05).

Article Snippet: The NE inhibitor, fragment of elafin, was from Anaspec (Fremont, CA).

Techniques: Inhibition, Activation Assay, Expressing, Western Blot, Phospho-proteomics

Journal: Journal of cellular immunology

Article Title: Evaluation of Neutrophil Elastase Inhibitors as Potential Therapies for ELANE Associated Neutropenia

doi: 10.33696/immunology.6.208

Figure Lengend Snippet: Inhibitors. The manufacturers and working concentrations of the inhibitors used in this study are listed.

Article Snippet: MK0339 NE inhibitor was provided by Merck & Co. (Kenilworth, NJ, USA).

Techniques: Concentration Assay, Control

Journal: Journal of cellular immunology

Article Title: Evaluation of Neutrophil Elastase Inhibitors as Potential Therapies for ELANE Associated Neutropenia

doi: 10.33696/immunology.6.208

Figure Lengend Snippet: Healthy volunteer and patient derived CD34+ cells were differentiated for 14 days in the presence or absence of inhibitors. The resultant cells were labeled with antibodies to CD14, CD66b, CD11b, and CD15 surface markers and analyzed using flow cytometry. A. Representative experiment histograms are shown. The proportion of CD14 + /CD66b + and CD11b + /CD15 + positive cells in quadrant 2 are indicated. B. Graphical representation of the percentage of CD66b + /CD14 + and CD11b + /CD15 + cellular subsets of the patients' cells after the addition of NE inhibitors. For each individual experiment, the percentage of cells with a mature phenotype after addition of inhibitor was divided by the percentage measured when only the vehicle control was added and this ratio was plotted. Data from 5 different patients, represented in at least two different experiments. Each individual patient has a different symbol. C. Cell cytospins stained with Kwik-Diff (eosin/methylene blue) were imaged using a Nikon digital camera. Cell differentiation was evaluated at 400x magnification by light microscope. Representative experiments showing the effect of MK0339 and brensocatib inhibitors on healthy volunteer and patient cells are shown.

Article Snippet: MK0339 NE inhibitor was provided by Merck & Co. (Kenilworth, NJ, USA).

Techniques: Derivative Assay, Labeling, Flow Cytometry, Control, Staining, Cell Differentiation, Light Microscopy

Journal: Journal of cellular immunology

Article Title: Evaluation of Neutrophil Elastase Inhibitors as Potential Therapies for ELANE Associated Neutropenia

doi: 10.33696/immunology.6.208

Figure Lengend Snippet: Molecular docking simulations for NE inhibitors were conducted using the MOE software package. The simulations utilized ELANE G214R, P139L, and wild-type models of neutrophil elastase. The images captured from the simulations display the binding sites of the inhibitors MK0339, sivelestat, GW311616, and BAY-678 with the wild-type NE. The inhibitor molecules are highlighted in color and indicated by arrows.

Article Snippet: MK0339 NE inhibitor was provided by Merck & Co. (Kenilworth, NJ, USA).

Techniques: Software, Binding Assay

Journal: Journal of cellular immunology

Article Title: Evaluation of Neutrophil Elastase Inhibitors as Potential Therapies for ELANE Associated Neutropenia

doi: 10.33696/immunology.6.208

Figure Lengend Snippet: Molecular docking simulation analysis. Molecular docking simulations for NE inhibitors were conducted using the MOE software package. The simulations utilized ELANE G214R, P139L, and wild-type models of neutrophil elastase. The stability of each inhibitor-protein complex was assessed using the free energy of binding (S score in kcal/mol), providing an approximation of binding stability.

Article Snippet: MK0339 NE inhibitor was provided by Merck & Co. (Kenilworth, NJ, USA).

Techniques: Software, Binding Assay