Investigating the Link between Alpha-1 Antitrypsin and Human Neutrophil Elastase in Bronchoalveolar Lavage Fluid of COVID-19 Patients

Abstract

Neutrophils play a pathogenic role in COVID-19 by releasing Neutrophils Extracellular Traps (NETs) or human neutrophil elastase (HNE). Given that HNE is inhibited by α1-antitrypsin (AAT), we aimed to assess the content of HNE, α1-antitrypsin (AAT) and HNE-AAT complexes (the AAT/HNE balance) in 33 bronchoalveolar lavage fluid (BALf) samples from COVID-19 patients. These samples were submitted for Gel-Electrophoresis, Western Blot and ELISA, and proteins (bound to AAT or HNE) were identified by Liquid Chromatography-Mass Spectrometry. NETs' release was analyzed by confocal microscopy. Both HNE and AAT were clearly detectable in BALf at high levels. Contrary to what was previously observed in other settings, the formation of HNE-AAT complex was not detected in COVID-19. Rather, HNE was found to be bound to acute phase proteins, histones and C3. Due to the relevant role of NETs, we assessed the ability of free AAT to bind to histones. While confirming this binding, AAT was not able to inhibit NET formation. In conclusion, despite the finding of a high burden of free and bound HNE, the lack of the HNE-AAT inhibitory complex in COVID-19 BALf demonstrates that AAT is not able to block HNE activity. Furthermore, while binding to histones, AAT does not prevent NET formation nor their noxious activity.

Keywords: COVID-19; Liquid Chromatography Mass Spectrometry (LC-MS); Neutrophils Extracellular Traps (NETs); alpha-1-antitrypsin (AAT); broncho-alveolar lavage (BAL); histones; human neutrophil elastase (HNE); lung; neutrophils.

Figure 1

Panel (A). Western blotting with anti-AAT antibody. Lanes 1–4: COVID-19 samples not showing the complex (80 kDa band) upon addition of exogenous HNE. Lanes 5–8: BOS samples showing the complex (80 kDa band) upon addition of exogenous HNE. Lane 9: complex generated “in vitro” upon incubation of the HNE and AAT standard proteins. Panel (B). Western blotting with anti-AAT and anti-HNE antibodies. Lane 1: free HNE standard protein. Lanes 2–9: different COVID-19 samples showing an intensity increase of the HNE band upon incubation with scalar amounts of exogenous HNE.

Figure 2

Western blotting with anti-HNE antibody. Five different COVID-19 samples show the presence of proteins in the range between 130 and 280 kDa which complexed with HNE.

Figure 3

12.5% SDS-PAGE (on the left) showing the protein profile of the BALf samples considered. The gel on the right shows an expansion (obtained by running the samples on an 8% gel) of the region marked with a dotted line in the gel on the left. The bands excised, digested with Trypsin, and submitted to protein identification by LC-MS are those indicated by the letters A to G in both gels.

Figure 4

Panel (A). Quantification of CitH3 in neutrophils activated towards NETosis using PMA (100 nM) with or without AAT (500 µg/mL) by ELISA assay after 4 h of incubation. Data are represented as mean of three independent replicates ± standard deviation. *** p < 0.01 vs. CTR; ^ p < 0.01 vs. PMA. Panel (B). Confocal microscopy images of neutrophils activated towards NETosis with PMA adding fluorescent AAT (green). DNA was labeled with DAPI and HNE with specific antibody (red). Scale bar = 50 µm.