key: cord-0807884-nu68as3a
authors: Seren, Seda; Derian, Lohann; Keleş, Irem; Guillon, Antoine; Lesner, Adam; Gonzalez, Loïc; Baranek, Thomas; Si-Tahar, Mustapha; Marchand-Adam, Sylvain; Jenne, Dieter E.; Paget, Christophe; Jouan, Youenn; Korkmaz, Brice
title: Proteinase release from activated neutrophils in mechanically ventilated patients with non-COVID-19 and COVID-19 pneumonia
date: 2021-04-29
journal: Eur Respir J
DOI: 10.1183/13993003.03755-2020
sha: 6662d0182bce2680f38d2b5777d3fe039ee013ee
doc_id: 807884
cord_uid: nu68as3a

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

Streptococcus pneumoniae, Mycoplasma pneumoniae or Streptococcus pneumoniae and Aspergillus fumigatus.

Flow cytometry analysis of the cellular content of ETAs showed that neutrophils (live CD45 + /CD14 − / CD16 + cells) accounted for the vast majority of leukocytes in the airways of pneumonia-related ARDS and, of note, no significant difference between the two groups was found (non-COVID-19 ARDS: 95±0.7%; COVID-19 ARDS: 94±0.9%, p=0.16) (figure 1a). ETA neutrophils from the two groups expressed similar amount of surface activation marker CD16 (mean fluorescence intensity (MFI) for non-COVID-19 ARDS: 19930±2617; MFI for COVID-19 ARDS: 20057±2112, p=0.7340; Mann-Whitney test). Myeloperoxidase (MPO), a marker of neutrophil activation, was detected in all ETA samples of non-COVID-19 and COVID-19 ARDS patients, with mean concentrations of 1148±102 ng·mL −1 and 1218±111 ng·mL −1 respectively, and no significant difference in concentration between the two groups ( p=0.63) (figure 1b). CatC activity was detected in ETA samples of 14 non-COVID-19 ARDS patients (93%; 2.6±0.8 ng·mL −1 ) and 14 COVID-19 ARDS patients (82%; 8±2.8 ng·mL −1 ) with no significant difference in concentration between the two groups ( p=0.39) (figure 1c). CatC activity was assayed using a CatC-selective FRET substrate [12] in the presence or absence of a selective CatC inhibitor (figure 1d). The 20-kDa heavy chain of CatC, representing the fully processed proteolytically active subunit of the proteinase, was detected by Western blotting using an antibody against CatC in a subset of the non-COVID-19 ARDS group ETA samples. When measured activity levels were low (figure 1d), this was reflected by an absence of the heavy chain band in the corresponding blot (figure 1e). Detection of proteolytically active CatC indicates the presence of activated neutrophils with granule release and consequently NSPs at inflammatory sites.

The selective and specific FRET substrates designed for each NSP [13] allowed their identification as active proteinases in ETA from patients (figure 1f-h). A reduction of NSP activities was achieved in our samples with purified alpha-1-antitrypsin, a natural serpin inhibitor of NSPs in human plasma. In this way, we were able to exclude the presence of active NSPs as a component of alpha-2-macroglobulin complexes, which encage, but still contain, the active proteinases. NSPs were detected in most samples also containing mature CatC (72%). NSP activity was detected in ETA of 12 non-COVID-19 ARDS patients (80%) and 14 COVID-19 ARDS patients (82%). We estimated the concentration of active NSPs from the rate of substrate hydrolysis using purified NSPs (non-COVID-19 ARDS group: PR3 14.8±4.4 ng·mL −1 , NE 29.9±9.6 ng·mL −1 , CatG 23.6±8.2 ng·mL −1 ; COVID-19 ARDS group: PR3 10.2±2.9 ng·mL −1 , NE 16±6.2 ng·mL −1 , CatG 23.7±11.7 ng·mL −1 ). We found no significant difference in the total concentration of the three NSPs between the non-COVID-19 ARDS group and COVID-19 ARDS (68.4±19.7 ng·mL −1 of ETA and 49.9±20.2 ng·mL −1 of ETA, respectively, p=0.52). Individual analysis of the three NSPs did not reveal any difference between non-COVID-19 and COVID-19 groups (figure 1f-h). The variation observed in NSP concentrations could be explained by neutrophil counts in the airways, their degranulation status and the level of active extracellular inhibitors. Last, we explored circulating NSP concentration in the serum of non-COVID-19 and COVID-19 patients and compared them to healthy donors (n=10). Because of huge amounts of serpins in sera, it was not possible to detect any proteolytic activity using NSP selective FRET substrates, despite their high sensitivity. We used an ELISA (enzyme-linked immunosorbent assay) that was particularly sensitive for detection of free NE and complexed NE with alpha-1-antitrypsin. There was no significant difference in NE serum concentration between non-COVID-19 and COVID-19 groups (280±29 ng·mL −1 of serum and 273±32 ng·mL −1 of serum, respectively, p=1) as assayed by ELISA (n=10) (figure 1i). However, NE concentration in both groups was significantly higher compared to healthy donors (median (interquartile range) age was 59 (54-63.5) years; male/female ratio was 2/1, n=10) (107±3.5 ng·mL −1 of serum; p<0.001, Kruskal-Wallis test).

Specific identification and detection of proteolytically active NSPs in acute inflammatory lung diseases is an important starting point to infer the simultaneous inhibition of multiple NSPs as a useful and effective therapeutic approach. By analysing ETA samples, we present strong circumstantial evidence for the involvement of NSPs in pneumonia-driven ARDS, in both non-COVID-19 and COVID-19 patients. It should, however, be kept in mind that pneumonia-driven ARDS is an alveolar process and that ETA sampling does not precisely reflect the unfolding inflammatory process in the alveolar compartment. Despite these technical limitations, our study strongly suggests that COVID-19 ARDS is associated with the release of biologically active NSPs into the small airways at a comparable level as observed in non-COVID-19 ARDS. It is thought that elevated levels of NSPs overwhelm the endogenous shield of natural inhibitors. In addition, we identified active CatC, the critical enzyme stored together with mature NSPs in primary granules of neutrophils, in ETA samples of non-COVID-19 and COVID-19 ARDS patients. Although no major difference between the two groups was found, we cannot exclude a difference below the limited statistical power of the study due to small numbers of patients. Since our observations have been made on a limited number of patients and because neutrophils may be involved in different detrimental processes in COVID-19 pathophysiology (including diffuse alveolar damage, thrombosis and Taken together, our data strongly encourage further mechanistic studies with regard to the precise contribution of NSPs in pneumonia-driven ARDS and strongly support initiatives to evaluate the therapeutic potential and efficacy of CatC inhibitors in COVID-19 to eliminate NSPs already at an early phase of the disease [4] . In this regard, the outcome of an ongoing clinical trial with brensocatib, a reversible nitrile CatC inhibitor approved in a phase 2 clinical study [14] , is eagerly awaited by health professionals, scientists and currently affected patients (STOP-COVID19, Superiority Trial of Protease Inhibition in COVID-19, EudraCT number 2020-001643-13 [4] ). Brensocatib treatment may help prevent irreversible pulmonary failure of COVID-19 patients with high level of active NSPs. Clinical trial results from the STOP-COVID-19 study will help us to better understand NSP-dependent pathophysiology of neutrophils in COVID-19. 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. 

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Conflict of interest: S. Seren has nothing to disclose. L. Derian has nothing to disclose. I. Keleş has nothing to disclose. A. Guillon has nothing to disclose. A. Lesner has nothing to disclose. L. Gonzalez has nothing to disclose. T. Baranek has nothing to disclose. M. Si-Tahar has nothing to disclose. S. Marchand-Adam has nothing to disclose. D.E. Jenne has nothing to disclose. C. Paget has nothing to disclose. Y. Jouan has nothing to disclose. B. Korkmaz has been paid for the time spent as a committee member for advisory boards (INSMED), other forms of consulting (Neuprozyme Therapeutics Aps (Denmark), Santhera Pharmaceuticals (Switzerland)), symposium organisation (INSMED) and travel support, lectures or presentations, outside the submitted work. Support Statement: This work was supported by the "Ministère de l'Enseignement Supérieur et de la Recherche", the "Région Centre Val de Loire" (Project Pirana) and "National Science Center Poland" (UMO 2014/15/B/ST5/05311) granted to A. Lesner. Funding information for this article has been deposited with the Crossref Funder Registry.