Proteinase release from activated neutrophils in mechanically ventilated patients with non-COVID-19 and COVID-19 pneumonia

Abstract

COVID-19 ARDS is associated with release of biologically active neutrophil elastase-related proteinases to the airways and blood at a comparable level to non-COVID ARDS https://bit.ly/3nihveh

FIGURE 1

Neutrophils and proteolytically active proteinases in endotracheal aspirates (ETAs). Schematic of the workflow is shown above. ETAs collected from mechanically ventilated non-COVID-19 acute respiratory distress syndrome (ARDS) and coronavirus disease 2019 (COVID-19) ARDS patients from intensive care unit were weighed and incubated in PBS (5 mL·g⁻¹) with 1 mM dithiothreitol (DTT) for 30 min at 4°C under gentle agitation. After centrifugation, supernatants were collected and then concentrated five times before total protein quantification by the BCA or Bradford assay. Cell pellets were filtered through a 100 µm cell strainer. Red blood cells were removed using a red blood cell lysis buffer and then cell suspensions from ETAs were passed through a 40 µm cell strainer prior to staining for flow cytometry. a) Cells were stained with antibodies against surface markers (CD45, CD14 and CD16) and viability dye. Neutrophils were defined as live CD45⁺ CD14⁻ CD16⁺ cells. Neutrophil relative proportion in ETAs of all non-COVID-19 ARDS and COVID-19 ARDS patients is represented as percentage of live leukocytes (CD45⁺ cells). Individuals are depicted. The data represent mean±sem. Mann–Whitney test. b) Quantitative detection of myeloperoxidase (MPO) in ETA supernatants (dilution: 1/500) from 14 non-COVID-19 ARDS and 14 COVID-19 ARDS patients using a human MPO Elisa kit (DMYE00B, R&D Systems). Individuals are depicted. The data represent mean±sem. Mann–Whitney test. c) The concentration of active cathepsin C (CatC) was estimated by comparison of the rate of hydrolysis of FRET (fluorescence resonance energy transfer) substrate by ETA supernatants of all non-COVID-19 ARDS and COVID-19 ARDS patients and recombinant CatC. Each point represents data of two independent experiments, both in duplicate, from an individual subject. Individuals are depicted. The data represent mean±sem. Mann–Whitney test. d) The CatC activity in five times concentrated ETA supernatants (4 µg protein) was measured spectrofluorometrically at 460 nm with or without the nitrile inhibitor (L)-Thi-(L)-Phe-CN (0.5 µM final, 20 min incubation at 37°C) using Thi-Ala(Mca)-Ser-Gly-Tyr(3-NO₂)-NH₂ (20 µM final) as selective FRET substrate. The residual activities were inhibited by the CatC inhibitor (L)-Thi-(L)-Phe-CN which demonstrate the presence of CatC activity in samples. Inhibition experiments were performed with all ETA samples used in the study. Results with ETA samples from 13 non-COVID-19 ARDS are shown. Data represent the mean±sd of two independent experiments, both performed with duplicates of the same sample. Similar results were obtained with ETA supernatants of all COVID-19 ARDS patients. FU: fluorescence unit. e) Western blotting (WB) of concentrated ETA supernatants (30 µg of protein) from 13 non-COVID-19 ARDS (no 1–13) using an anti-CatC antibody (sc-74590, 1:1000, Santa Cruz Biotechnology) shows the presence of the CatC heavy chain. Similar results were obtained with ETA supernatants of all non-COVID-19 ARDS and COVID-19 ARDS patients in three independent experiments. f) The proteinase 3 (PR3) activity in ETA supernatants was measured at 420 nm with or without the PR3 inhibitor Ac-PYDA^P(O-C₆H₆-4-Cl)₂ (0.5 µM final, 20 min incubation at 37°C) using ABZ-VAD(nor)VADYQ-EDDnp (20 µM final) as a substrate. g) CatG activity in ETA supernatants was measured in the presence or absence of alpha-1-antichymotrypsin using ABZ-TPFSGQ-EDDnp (20 µM final) as a substrate. h) The neutrophil elastase (NE) activity in ETA supernatants was measured with or without NE inhibitor EPI-hNE-4 (0.5 µM final, 20 min incubation at 37°C) using ABZ-APEEIMRRQ-EDDnp as a substrate. The residual activities in ETA samples were inhibited by selective NSP inhibitors demonstrating the specificity of the FRET assays utilised. Inhibition experiments were performed with all ETA samples used in the study. Each point represents data of two independent experiments, both in duplicate, from an individual subject. Individuals are depicted. The data represent mean±sem. Mann–Whitney test. i) Quantitative detection of circulating NE. A human NE ELISA kit (HK319, HycultBiotech) was used for the quantitative detection of NE in sera (dilution: 1/100) from 10 non-COVID-19 ARDS and 10 COVID-19 ARDS patients. Individuals are depicted. The data represent mean±sem. Kruskal–Wallis test. ns: nonsignificant.