key: cord-0742583-7qsixuqt
authors: Fisher, Jane; Mohanty, Tirthankar; Karlsson, Christofer; Khademi, S. M. Hossein; Malmström, Erik; Frigyesi, Attila; Nordenfelt, Pontus; Malmstrom, Johan; Linder, Adam
title: Proteome profiling of recombinant DNase therapy in reducing NETs and aiding recovery in COVID-19 patients
date: 2021-06-15
journal: Mol Cell Proteomics
DOI: 10.1016/j.mcpro.2021.100113
sha: 62c1ba1baf5480f93dc7bcd317a51e43d548e07a
doc_id: 742583
cord_uid: 7qsixuqt

Severe COVID-19 can result in pneumonia and acute respiratory failure. Accumulation of mucus in the airways is a hall mark of the disease and can result in hypoxemia. Here, we show that quantitative proteome analysis of the sputum from severe COVID-19 patients reveal high levels of neutrophil extracellular trap(s) (NETs) components, which was confirmed by microscopy. Extracellular DNA from excessive NET formation can increase sputum viscosity and can lead to acute respiratory distress syndrome (ARDS). Recombinant human DNase (rhDNase/Pulmozyme) has been shown to be beneficial in reducing sputum viscosity and improve lung function. We treated 5 COVID-19 patients presenting acute symptoms with clinically approved aerosolized Pulmozyme. No adverse reactions to the drug were seen, and improved oxygen saturation and recovery in all severely ill COVID-19 patients was observed after therapy. Immunofluorescence and proteome analysis of sputum and blood plasma samples after treatment revealed a marked reduction of NETs and a set of statistically significant proteome changes that indicate reduction of haemorrhage, plasma leakage and inflammation in the airways, and reduced systemic inflammatory state in the blood plasma of patients. Taken together, the results indicate that NETs contribute to acute respiratory failure in COVID-19 and that degrading NETs may reduce dependency on external high flow oxygen therapy in patients. Targeting NETs using rhDNase may have significant therapeutic implications in COVID-19 disease and warrants further studies.

COVID-19, the pandemic disease caused by the novel coronavirus SARS-CoV2 (previously named 2019-nCoV) causes symptoms with severity ranging from a mild cold to severe pneumonia and acute respiratory distress syndrome (ARDS) that in some cases is fatal [1] [2] [3] . The World Health Organization estimates that 15% of patients will have severe disease, 5% will have critical disease and 3-4% will succumb to the diseae 4 . Patients with severe COVID-19 frequently develop ARDS and respiratory failure, characterized by hypoxemia, neutrophilia, pulmonary neutrophil infiltration, fibrin deposition, build-up of thick mucus in the bronchi and bronchiectasis 5 . Severely ill patients exhibit laboured breathing and often require oxygen therapy through high flow nasal oxygenation (HFNO), mechanical ventilation or extracorporeal membrane oxygen therapy (ECMO) 6 . However, these strategies have limitations due to harmful side effects 7 and an insufficient supply of ventilators 8 . ARDS in COVID-19 patients is characterised by damaged alveoli, edema, haemorrhage and intraalveolar fibrin deposition 9 . This causes a hazy appearance of blood vessels and airway structures when viewed using computerized tomography (CT) imaging and this phenotype is termed glass ground opacity (GGO) 10 . Haemorrhage, plasma leakage and pulmonary neutrophil infiltration 3, 9 can cause the build-up of gelatinous and highly viscous sputum, which in turn produces the GGO phenotype in COVID-19 lungs 5 . So far, molecular composition of sputum from Covid-19 patients has remained uncharacterized. However, similar symptoms are seen in lungs of patients with ARDS and Cystic fibrosis (CF), where neutrophil influx, acute phase plasma proteins and inflammatory cytokines are present in sputum 11-13 . Sputum in ARDS and CF is highly complex and apart from proteins also contains abundant extracellular DNA, which causes mucus thickening. In fact, previous reports have shown extracellular DNA to increase mucus viscosity by 30% in CF 14 and inability to clear sputum from airways can lead to exacerbations and respiratory hypoxemia 15 . During ARDS and CF, Neutrophils can directly contribute to the extracellular DNA pool by forming neutrophil extracellular traps (NETs) 16, 17 . NETs J o u r n a l P r e -p r o o f Proteome profiling in COVID-19 after DNase therapy consist of extracellular DNA bound to neutrophil granule proteins, and are released in response to bacteria 18 as well as some viruses 19 . DNA decondensation preceding NET formation requires myeloperoxidase (MPO), neutrophil elastase (NE) and peptidyl arginine deiminase IV (PADI-4) activity 20 . PADI-4 is known to catalyze arginine residues to citrulline in histones 21 and other granule proteins during NET formation 22, 23 . NETs are hypothesized to aid the immune response by immobilizing and neutralizing virus particles 19, 20 . Knockout of PADI-4 did not worsen experimental influenza 24 , suggesting that NETs are not always an essential part of the immune response to viral infections. NETs can be cytotoxic to endothelial and lung epithelial cells 25 and can induce clot formation leading to vascular occlusion in the lungs 26 , suggesting that a dysregulated NET response in the lungs can lead to significant damage. It has been hypothesized that NETs may play a role in COVID-19 27, 28 and markers of NETs have been detected in the plasma of COVID-19 patients 29 .

In CF, NETs can be degraded using deoxyribonuclease (DNase I). Preclinical studies have suggested that removal of NETs using DNase is beneficial in both bacterial 30 and viral 31, 32 diseases. Production of highly viscous sputum may cause ARDS in COVID-19. Accumulation of thick sputum in the airways can interfere with the gaseous exchange, which in turn leads to hypoxemia 33 , increased use of mechanical ventilation and an increased risk of mortality. Therefore, improving mucus clearance from airways by altering sputum viscosity may improve pulmonary oxygenation and prevent development of ARDS. This strategy may also reduce dependency on mechanical ventilation and reduce the risk of mortality during COVID-19. Recombinant human DNase I (rhDNase) could potentially be used to target dysregulated NET formation in severe COVID-19. rhDNase (Pulmozyme) is currently used safely in humans to reduce mucus thickness in cystic fibrosis 34 . However, the current understanding of alterations in the composition of sputum and blood plasma proteome during SARS-CoV2 pathogenesis remains limited.

In this study we applied SWATH-like data independent acquisition mass spectrometry 35 (DIA-MS) to examine the sputum and blood plasma proteome from COVID-19 patients. We found J o u r n a l P r e -p r o o f neutrophil/NET-derived proteins, including neutrophil granule proteins and citrullinated proteins, and acute phase proteins associated with exaggerated inflammation in sputum.

Immunofluorescence analysis of sputum from COVID-19 revealed the presence of NETs in the sputum that could be degraded using DNase I ex vivo. Further, to gain preliminary insights into the action of rhDNase in improving respiratory function, a small cohort of severely ill COVID-19 patients were treated with rhDNase followed by molecular characterization of blood plasma and sputum using immunofluorescence and proteomics analysis. 

The sample collection was approved by the Lund University local ethics committee (application number 2016/39) and was in accordance with the ethical principles in the Helsinki declaration.

Informed consent was collected from all participants or next of kin. We enrolled ten patients in the study from March 17 to April 12, 2020. The included patients were admitted to the Clinic for Infectious Diseases in Lund with confirmed COVID-19 by positive SARS-CoV2 revere-transcriptasepolymerase-chain-reaction assay and a need for respiratory support to maintain an oxygen saturation >93%. Patients who were treated with rhDNase were followed with serial sampling until hospital discharge. Demographic and clinical data were collected retrospectively from the patients' charts. Venous EDTA-blood (K2EDTA, BD vacutainer, 10ml) was collected once or twice prior to rhDNase treatment and once-daily following rhDNase treatment. Sputum was collected whenever it was possible by spontaneous production (coughing). Not all patients were able to expectorate sputum, and therefore they were excluded from sputum NET analyses where relevant. Platelet poor plasma was collected by centrifuging EDTA blood @1800g for 10 minutes at room temperature.

Sputum samples were collected in 70 ml multi-purpose polypropylene sterile containers without any additives (Sarstedt, Germany). All samples were processed within 4 hours after collection from patients. The interval between processing times and freezing of samples were limited to a maximum of15 minutes for plasma and 30 minutes for sputum to minimise variability.

Blood and sputum were collected from four donors who were not exhibiting any respiratory symptoms and therefore were assumed to be SARS-CoV2 negative. We cannot rule out asymptomatic infections in these donors. Collection of blood from healthy donors was approved by the Lund University local ethics committee (application number 2013/728).

Proteome profiling in COVID-19 after DNase therapy Treatment All patients were given standard clinical care for their condition. Three SARS-CoV2 positive patients were analysed for sputum NETs but were not treated with rhDNase. Five SARS-CoV2 positive patients (referred to as patients TP 1-5) treated with rhDNase (Pulmozyme; Roche), administered by the decision of the treating physician as "off-label" use. Four of these patients (TP 1-4) were able to expectorate sputum before and after treatment and were analysed for NETs. Treatment with rhDNase was given via nebuliser at a dose of 2.5 mg twice daily until the treating physician's decision to stop treatment. All patients were treated with oxygen therapy either by conventional oxygen therapy (COT) or high flow nasal oxygen (HFNO) therapy at time of treatment start. The intervention was not randomised, and patients and clinicians were not blinded.

Sputum sample from a COVID patient was treated with 10 units of recombinant human DNase I (Abcam) for 10' at 37 degrees Celsius. An aliquot of the same sample was treated the same way but without addition of DNase I. Samples were cytocentrifuged and then prepared for immunostaining as described below.

Estimated mean arterial pressure (MAP) was calculated by doubling the diastolic pressure and adding the systolic pressure and dividing this sum by 3. The fraction of inspired oxygen (FiO 2 ) when patients were receiving conventional oxygen therapy (COT) via nasal cannula or face mask was estimated by multiplying the oxygen flow rate by 0.04 and adding this number to 0.2 36 . When patients were receiving high flow nasal oxygen (HFNO) therapy, the FiO 2 was estimated by the oxygen percentage set on the blender. Because arterial oxygen partial pressure (PaO2)/FiO 2 was not measured in these patients, the SpO 2 /FiO 2 ratio was calculated as a surrogate 37 . SpO 2 was the saturation of oxygen measured by pulse oximetry. For 3D super-resolution imaging, samples were stained with rabbit-anti-human neutrophil elastase antiserum and AF-568 conjugated goat-anti-rabbit secondary antibody. DNA was stained with 5 µM DRAQ5 for 30 minutes at room temperature, and coverslips were mounted with ProLong Gold antifade reagent (Life Technologies).

Some samples were prepared for same-day analysis. The protocol for NET analysis was followed as above with some changes. Fixation was done for a minimum of 30 minutes at 4 °C. Blocking was done for a minimum of 15 minutes at 37 °C. Primary and secondary antibody incubation was done for a minimum of 15 minutes at room temperature. Samples were washed as normal, and coverslips were mounted with a drop of mounting media with DAPI. Clear nail polish was applied to the edges of the coverslip and was then allowed to dry. Samples were imaged directly. 

All unstitched frames from each sample were quantified using the NETQUANT app (version 1.3) in MATLAB (version 2019b) 38 . The software uses thresholds for NET criteria that are set by the user, and for the analysis applied here, the thresholds were: cell area fold increase 3.50; nuclei deformation 0.30; DNA/NET area 0.8 or 2.0. Elastase staining in the samples was heterogeneous, likely due to varying amounts of neutrophil activation between patients, making it necessary to apply two different segmentation settings for the elastase channel. The "Global" option applies Otsu's method 39 , where a segmentation threshold is selected that minimises the intraclass variance of black and white pixels. The "Adaptive" option uses Bradley's method to calculate a locally adaptive threshold using local first-order statistics around each pixel(Bradley and Roth, 2007). For the DNA channel, adaptive segmentation was used with a sensitivity of 0.2. Representative images were processed in Fiji.

J o u r n a l P r e -p r o o f All super-resolution images were collected with an N-SIM E system on Nikon Ti-E inverted microscope equipped with a Plan Apochromat Lambda 100X/1.49 (magnification/NA). AF-594 was excited with the 561 nm line from a laser source and collected with an N-SIM561 filter. DRAQ5 was excited with the 640 nm line from a laser source and collected with an N-SIM640 filter. Z-series optical sections were collected with a step-size of 0.3 microns. Images were acquired with a Hamamatsu Orca Flash 4.0 sCMOS camera controlled with NIS Elements AR software. The SIM images were reconstructed with the NIS-elements AR algorithm for reconstruction.

To liquefy the sputum, all sputa were treated with 15mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) (Sigma) for 10 minutes at room temperature. These were then centrifuged at 500g for 10 min at room temperature to obtain a supernatant and pellet. The pellet was resuspended in 100µl 0.2% RapiGest SF surfactant (Waters) and boiled for 10 minutes and cooled on ice for 10 minutes. An equal volume of 8M urea in 0.1M ammonium bicarbonate (Sigma) solution was added to the samples. BCA (Pierce) was then performed on the samples to estimate protein concentration, and 50µg of protein was taken for digestion.

Blood from EDTA tubes was processed by centrifugation for 10 minutes at 500g at room temperature to obtain the buffy coat. The supernatant was taken into fresh tubes and spun for 10 minutes at 2000g to remove platelets. 100µl of plasma was diluted 1:10 by adding 900µl of 8M urea-0.1M ammonium bicarbonate solution and stored at -20°C. 10 µl of the diluted plasma was digested.

Proteins were reduced with 5mM TCEP, pH 7.0 for 45 min at 37 °C, and alkylated with 25 mM iodoacetamide (Sigma, USA) for 30 min followed by dilution with 100 mM ammonium bicarbonate to a final urea concentration below 1.5 M. Proteins were digested by incubation with trypsin (1/100, w/w, Sequencing Grade Modified Trypsin, Porcine, Promega) for at least 9h at 37 °C. Digestion was stopped using 5% trifluoracetic acid (Sigma) to pH 2-3. The peptides were cleaned up by C18 reversed-phase spin columns as per the manufacturer's instructions (Silica C18 300 Å Columns, J o u r n a l P r e -p r o o f Harvard Apparatus, USA). Solvents were removed using a vacuum concentrator (Genevac, miVac) and were resuspended in 50 µl HPLC-water (Fisher Chemical) with 2% acetonitrile, 0.2% formic acid (Sigma). Samples were spiked with iRT peptides prior to MS analysis.

All peptide analyses were performed on a Q Exactive HF-X mass spectrometer (Thermo Fisher Scientific) connected to an EASY-nLC 1200 ultra-high-performance liquid chromatography system bacterial infections (n=3). Quantitative DIA mass spectrometry analysis reveals that the sputum proteome is highly complex, variable and comprises of more than 2000 unique proteins (Figure 1 A) .

In COVID-19, the most abundant proteins were both subtypes of immunoglobulin A and mucins, followed by blood plasma proteins such as albumin, leukocyte proteins and inflammatory/antiviral response proteins such as interferon-induced proteins (Supplementary table-1) . Figure   1B ). PADI-4 or PADV is a neutrophil enriched nucleus enzyme that catalyzes citrullination of histones and neutrophil proteins prior to NET release 22, 23 . Interestingly, several neutrophil proteins and histones were citrullinated in COVID-19 (Fig. S1.) , possibly as a result of the detectable levels of PADI-4 or PADV 47 (Figure 1B) . Taken together, these results indicate that the sputum is inflammatory J o u r n a l P r e -p r o o f active and that there are high levels of neutrophil and a substantial degree of NET-related proteins in COVID-19 sputum.

Discovery of neutrophil proteins, PADI enzymes, citrullinated neutrophil proteins and histones, all of which are associated with NET formation, prompted us to investigate whether NETs are present within the respiratory system of patients with COVID-19. We collected sputum from SARS-CoV2 positive patients with different disease severities admitted to the clinic for infectious diseases at Skåne University hospital in Lund, Sweden. Only nine patients were able to expectorate sputum.

Using immunofluorescence microscopy against neutrophil elastase (NE) and DNA as described previously 30 , and in agreement with the mass spectrometry data, we readily found neutrophil infiltration and NETs in the sputum of COVID-19 patients (Figure 2 A) . Cells were found in various stages of NETosis ( Figure 2B and Supplementary Movie 1), ranging from early-phase NETs (Figure 2 B, bottom arrow) to large, diffuse, and completely extracellular NET structures (Figure 2 B (Fig. S2.) .

The DNase treated sample also showed a marked reduction in turbidity and viscosity upon visual inspection.

The marked reduction in turbidity and viscosity after DNase treatment indicates that aerosolized DNase could be used to degrade extracellular DNA, reduce sputum viscosity, and improve respiratory function. To test this notion, we treated 5 severely ill COVID-19 patients admitted to the clinic for infectious diseases at Skåne University hospital in Lund, Sweden with rhDNase. All patients were at the time of treatment required external oxygen therapy (baseline characteristics of the patients receiving rhDNase therapy are presented in Table 1 and Table S1 .). Conventional oxygen therapy (COT) or HFNO was administered to the patients prior to rhDNase therapy. Remarkably, within 4-15 days of rhDNase treatment none of the patients went on to require ICU admission or mechanical ventilation, and all were weaned off oxygen therapy. Oxygen demand for the four HFNO patients (patients 1-4, Figure 3A ) declined within 1-3 days after start of rhDNase administration and SpO 2 /FiO 2 ratio began to climb concurrently ( Figure 3B ). All patients receiving rhDNase treatment recovered.

To study the effect of rhDNase on sputum NETs, we analysed sputum NETs over time in 4 patients who produced sputum both before and after treatment using immunofluorescence and DIA-MS analysis. Representative images of sputum NETs before and after rhDNase treatment in each patient receiving HFNO therapy at treatment start are shown in Figure 4 . Quantification of immunofluorescence against NE and DNA revealed that prior to rhDNase treatment (0.5-1 days), the patients had 23-31% NET-forming cells (Figure 5 A) and with DNA size of 2000-6500px (Figure 5 B) . 59 .

NETs have been described to be formed in response to SARS-CoV-2 60 however underlying host molecular mechanisms that govern NET formation in COVID-19 have not been described in detail.

Increases in NET formation have been linked to severity and mortality in COVID-19 61, 62 . In line with J o u r n a l P r e -p r o o f previous findings, we also found increased NET formation prior to rhDNase therapy. This was gradually reversed following therapy and recovery in all patients was associated with reduced NETs in the sputum. Further studies are needed to evaluate if lowered NETs in sputum are a marker of recovery in COVID-19.

rhDNase treatment reduced sputum NETs within three days, this indicates that rhDNase can penetrate sputum at clinically relevant doses and therefore could be a viable therapeutic to target

NETs. DNase levels detected in the sputum during treatment varied over time and between patients, as observed in previous pharmacokinetic studies 63 . Plasma levels of DNase were undetectable by mass spectrometry before and after treatment start, likely due absent or low concentrations of DNase in the blood. Previous studies have also found that aerosolised rhDNase administration did not significantly increase plasma DNase levels 64, 65 . The patients were treated with Pulmozyme every 12 hours over several days without any observable adverse effects, demonstrating that the drug was well tolerated. Overall, our data indicate that rhDNase pharmacokinetics and mode of action in COVID-19 sputum are likely similar to healthy and cystic fibrosis sputum, suggesting that doses and administration frequencies currently recommended for cystic fibrosis can be used in future studies of rhDNase in COVID-19. At the same time, there are still remnants of neutrophil proteins and NETs in the sputum after treatment, which indicates that higher doses of rhDNase potentially could more efficiently remove all DNA. Following rhDNase treatment, all five patients receiving COT or HFNO therapy did not deteriorate further and did not require ICU care or a ventilator at any time. These patients were weaned from high-flow oxygen within four days of treatment start. Our results suggest that future clinical trials of rhDNase treatment should consider also including patients who are receiving oxygen therapy but do not yet require mechanical ventilation.

Although, rhDNase degraded NETs we observed an increase in S100 A8 and 9 that could originate from neutrophils. This implies that neutrophil influx occurs during the process of recovery. Further studies are needed to examine the role of neutrophils after treatment. rhDNase treatment resulted J o u r n a l P r e -p r o o f in an increase in eosinophil proteins in sputum. Eosinopenia is known to be associated with severe COVID 66 and the reappearance of eosinophils could indicate recovery. We also observed lower amounts of complement proteins and haemoglobin with treatment. Together, these data indicate that rhDNase treatment caused a cellular reorganization and lowered the levels of plasma proteins in the airways.

Improved oxygenation after rhDNase treatment was also associated with the reorganization of the blood plasma proteome. Hypoalbuminemia and thrombocytopenia are associated with poor outcomes in COVID-19 49, 67, 68 . Following treatment, albumin and platelets proteins increased in concentrations indicating the reversal of acute conditions. Proteins that are protective against thrombosis like SERPIND1 or heparin cofactor II and inter-alfa-trypsin inhibitor heavy chain 2 were also upregulated. This may indicate a novel protective effect against coagulopathies commonly associated with COVID-19. Levels of C-reactive protein (CRP), a known marker of disease severity in COVID-19, were also reduced following rhDNase treatment (see Fig. S6 ). Although this was not significant (p=0.07) a clear trend toward reduction was observed. A similar pattern of reduction for acute phase proteins, leukocyte proteins and hypoxia-upregulated protein 1. Taken together, the differentially regulated protein patterns in blood plasma described in the manuscript elucidate that improved oxygenation due to rhDNase therapy is associated with the lowering of hypoxemia, reduced inflammatory cells and acute phase response proteins.

Limitations in study design include small sample size, the lack of randomisation and controls, the lack of blinding at each analysis step, and the use of several concomitant medications such as chloroquine and low-molecular-weight heparin. Apart from this, sampling sputum posed a significant challenge as it could not be performed regularly due to variation in sputum production by patients.

Additionally, samples for microscopy had to be processed immediately, as freezing could introduce artefacts. This made storage, processing and performing batch analyses for microscopy very difficult.

For these reasons, we could only examine sputum NETs in a small cohort of patients.

We believe that our study provides important information to complement and validate previous ELISA-based reports of NETs in COVID-19 29 . No evidence has so far been provided about the presence of NETs in the lungs and their involvement in causing ARDS in COVID-19. Two recent reports also found DNase to be beneficial in COVID-19 69 also lowered GGO in lungs. The rhDNase treatment regimen of 2.5mg rhDNase twice daily followed in both manuscripts was similar to ours and were without any treatment-associated toxicities.

However, both reports did not address the presence of NETs through microscopy or ELISA-based methods in blood plasma, sputum or in the airways of the patients. One of the strengths of this manuscript is that we provide direct evidence showing the presence of intact NETs and NET-related proteins such as azurophilic granule-derived and citrullinated proteins in lungs, the main target organ of COVID-19. Secondly, we identified NETs as a source of extracellular DNA, which enhances sputum viscosity in severely ill COVID-19 patients and that rhDNase treatment reduced NETs in sputa of recovered patients. Furthermore, using mass spectrometry we were able to monitor the reorganization of sputum and blood plasma proteome suggesting reduced inflammatory proteins were associated with recovery after rhDNase treatment.

We realize that the reduction of inflammatory proteins, improved oxygenation and recovery presented here may also be independent of the rhDNase therapy. 

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We wish to thank Oscar André for enabling automated acquisition of widefield