Proteome Profiling of Recombinant DNase Therapy in Reducing NETs and Aiding Recovery in COVID-19 Patients
2021; Elsevier BV; Volume: 20; Linguagem: Inglês
10.1016/j.mcpro.2021.100113
ISSN1535-9484
AutoresJane Fisher, Tirthankar Mohanty, Christofer Karlsson, S. M. Hossein Khademi, Erik Malmström, Attila Frigyesi, Pontus Nordenfelt, Johan Malmström, Adam Linder,
Tópico(s)S100 Proteins and Annexins
Resumo•High levels of neutrophil extracellular traps (NETs) in the sputum of severe COVID-19 patients.•Recombinant human DNase decreased NETs in sputum.•Reduced NETs were associated with recovery and improved oxygenation.•Mass spectrometry analyses of plasma and sputum indicate resolution of inflammation. Severe coronavirus disease 2019 (COVID-19) can result in pneumonia and acute respiratory failure. Accumulation of mucus in the airways is a hallmark of the disease and can result in hypoxemia. Here, we show that quantitative proteome analysis of the sputum from severe patients with COVID-19 reveal high levels of neutrophil extracellular trap (NET) components, which was confirmed by microscopy. Extracellular DNA from excessive NET formation can increase sputum viscosity and lead to acute respiratory distress syndrome. Recombinant human DNase (Pulmozyme; Roche) has been shown to be beneficial in reducing sputum viscosity and improve lung function. We treated five patients pwith COVID-19 resenting 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 patients with COVID-19 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 hemorrhage, 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 recombinant human DNase may have significant therapeutic implications in COVID-19 disease and warrants further studies. Severe coronavirus disease 2019 (COVID-19) can result in pneumonia and acute respiratory failure. Accumulation of mucus in the airways is a hallmark of the disease and can result in hypoxemia. Here, we show that quantitative proteome analysis of the sputum from severe patients with COVID-19 reveal high levels of neutrophil extracellular trap (NET) components, which was confirmed by microscopy. Extracellular DNA from excessive NET formation can increase sputum viscosity and lead to acute respiratory distress syndrome. Recombinant human DNase (Pulmozyme; Roche) has been shown to be beneficial in reducing sputum viscosity and improve lung function. We treated five patients pwith COVID-19 resenting 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 patients with COVID-19 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 hemorrhage, 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 recombinant human DNase may have significant therapeutic implications in COVID-19 disease and warrants further studies. Coronavirus disease 2019 (COVID-19), the pandemic disease caused by the novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) (previously named 2019-nCoV, where "n" is for novel and "CoV" is for coronavirus), causes symptoms with severity ranging from a mild cold to severe pneumonia and acute respiratory distress syndrome (ARDS) that in some cases is fatal (1Huang C. Wang Y. Li X. Ren L. Zhao J. Hu Y. Zhang L. Fan G. Xu J. Gu X. Cheng Z. Yu T. Xia J. Wei Y. Wu W. et al.Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China.Lancet. 2020; 395: 497-506Abstract Full Text Full Text PDF PubMed Scopus (30846) Google Scholar, 2Liu K. Chen Y. Lin R. Han K. Clinical features of COVID-19 in elderly patients: A comparison with young and middle-aged patients.J. Infect. 2020; 80: e14-e18Abstract Full Text Full Text PDF PubMed Scopus (996) Google Scholar, 3Wu C. Chen X. Cai Y. Xia J. Zhou X. Xu S. Huang H. Zhang L. Zhou X. Du C. Zhang Y. Song J. 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PAD4-mediated neutrophil extracellular trap formation is not required for immunity against influenza infection.PLoS One. 2011; 6e22043Crossref PubMed Scopus (192) Google Scholar), 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 (25Saffarzadeh M. Juenemann C. Queisser M.A. Lochnit G. Barreto G. Galuska S.P. Lohmeyer J. Preissner K.T. Neutrophil extracellular traps directly induce epithelial and endothelial cell death: A predominant role of histones.PLoS One. 2012; 7e32366Crossref PubMed Scopus (873) Google Scholar) and can induce clot formation leading to vascular occlusion in the lungs (26Jiménez-Alcázar M. Rangaswamy C. Panda R. Bitterling J. Simsek Y.J. Long A.T. Bilyy R. Krenn V. Renné C. Renné T. Kluge S. Panzer U. Mizuta R. Mannherz H.G. 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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; Roche) is currently used safely in humans to reduce mucus thickness in CF (34Yang C. Chilvers M. Montgomery M. Nolan S.J. Dornase alfa for cystic fibrosis.Cochrane Database Syst. Rev. 2016; 4: CD001127PubMed Google Scholar). 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 MS (DIA-MS) (35Guo T. Kouvonen P. Koh C.C. Gillet L.C. Wolski W.E. Röst H.L. Rosenberger G. Collins B.C. Blum L.C. Gillessen S. Joerger M. Jochum W. Aebersold R. Rapid mass spectrometric conversion of tissue biopsy samples into permanent quantitative digital proteome maps.Nat. Med. 2015; 21: 407-413Crossref PubMed Scopus (271) Google Scholar) to examine the sputum and blood plasma proteome from patients with COVID-19. We found 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. Furthermore, to gain preliminary insights into the action of rhDNase in improving respiratory function, a small cohort of severely ill patients with COVID-19 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 transcription-polymerase 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;10 ml, BD Biosciences) 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 at 1800g for 10 min at room temperature. Sputum samples were collected in 70 ml multipurpose polypropylene sterile containers without any additives. All samples were processed within 4 h after collection from patients. The interval between processing times and freezing of samples was limited to a maximum of 15 min for plasma and 30 min for sputum to minimize 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). All patients were given standard clinical care for their condition. Three SARS-CoV2–positive patients were analyzed 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), 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 analyzed for NETs. Treatment with rhDNase was given via nebulizer 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 HFNO therapy at time of treatment start. The intervention was not randomized, and patients and clinicians were not blinded. Sputum sample from a patient with COVID was treated with 10 units of rhDNase (Abcam) for 10' at 37 °C. 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 later. Estimated mean arterial pressure was calculated by doubling the diastolic pressure and adding the systolic pressure and dividing this sum by three. The fraction of inspired oxygen (FiO2) when patients were receiving 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 (36Wilkins R.L. Stoller J.K. Kacmarek R.M. Egan's Fundamentals of Respiratory Care.9 Ed. Mosby Elsevier, St. Louis, MO2009Google Scholar). When patients were receiving HFNO therapy, the FiO2 was estimated by the oxygen percentage set on the blender. Because arterial oxygen partial pressure/FiO2 was not measured in these patients, the SpO2 (the saturation of oxygen as measured by pulse oximetry)/FiO2 ratio was calculated as a surrogate (37Rice T.W. Wheeler A.P. Bernard G.R. Hayden D.L. Schoenfeld D.A. Ware L.B. Comparison of the SpO2/FIO2 ratio and the PaO2/FIO2 ratio in patients with acute lung injury or ARDS.Chest. 2007; 132: 410-417Abstract Full Text Full Text PDF PubMed Scopus (512) Google Scholar). Sputum was immediately fixed with 4% paraformaldehyde (Sigma–Aldrich) in PBS (Sigma–Aldrich) at 4 °C for 1 h. About 10 μl of the fixed sputum mixture was diluted with 500 μl of PBS and cytocentrifuged for 10 min at 2000 rpm onto glass slides. Samples were permeabilized with 0.5% Triton X-100 (Sigma–Aldrich) for 20 s and then blocked with blocking buffer (5% goat serum [BioWest] with 0.05% Tween-20 [MP Biologicals] in PBS) at 37 °C for 30 min. Samples were stained with rabbit-antihuman NE antiserum (Dako; 1:500 dilution) in blocking buffer at 4 °C overnight, then stained with secondary Alexa Fluor (AF)-647–conjugated goat-anti-rabbit Fab2' antibody fragment (1:1000 dilution; Life Technologies) in blocking buffer at 37 °C for 1 h. Samples were washed three times with PBS and coverslips (Menzel-Glaser; #1.5 thickness) were mounted on the samples using mounting media with ProLong Gold antifade reagent with 4,6 diamidino-2-phenylindole (DAPI; Life Technologies) and cured overnight. For 3D super-resolution imaging, samples were stained with rabbit-antihuman NE antiserum and AF-568–conjugated goat-anti-rabbit secondary antibody. DNA was stained with 5 μM DRAQ5 for 30 min 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 aforementioned with some changes. Fixation was done for a minimum of 30 min at 4 °C. Blocking was done for a minimum of 15 min at 37 °C. Primary and secondary antibody incubation was done for a minimum of 15 min 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 widefield images were collected with a Nikon Ti-2 inverted microscope equipped with a 20×/0.75 or a 40×/0.95 (magnification/numerical aperture) objective and the Perfect Focus System for maintenance of focus over time. Fluorophores were excited with a Lumencor SpectraX light engine. AF-594 was excited with the 57- nm line from a 330 mW light-emitting diode source and collected with a DM593 dichroic mirror and a 624/40 nm emission filter. DAPI was excited with the 395-nm line from a 295 mW light-emitting diode source and collected with a DM409 dichroic mirror and a 447/60 nm emission filter. Images were acquired with a Nikon DS-Qi2 sCMOS camera controlled with NIS Elements AR software. Multiple stage positions were collected using a motorized piezo stage. Whole slide scanning was performed using NIS Elements JOBS to acquire 6 × 6 20× images to cover the whole sample circle. Frames were stitched using 5% overlap at the edges and automatic shading correction. All unstitched frames from each sample were quantified using the NETQUANT app (version 1.3) in MATLAB (version 2019b) (38Mohanty T. Sorensen O.E. Nordenfelt P. NETQUANT: Automated quantification of neutrophil extracellular traps.Front. Immunol. 2017; 8: 1999Crossref PubMed Scopus (25) Google Scholar). The software uses thresholds for NET criteria that are set by the user, and for the analysis applied here, the following are the thresholds: cell area fold increase 3.50; nuclei deformation 0.30; and DNA/NET area 0.8 or 2.0. Elastase staining in the samples was heterogeneous, likely because of 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 (39Otsu N. A threshold selection method from gray-level histograms.IEEE Trans. Syst. Man Cybern. 1979; 9: 62-66Crossref Google Scholar), where a segmentation threshold is selected that minimizes 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 (40Schindelin J. Fiji: an open-source platform for biological-image analysis.Nat. Methods. 2012; 9: 676-682Crossref PubMed Scopus (33774) Google Scholar). All super-resolution images were collected with an N-SIM E system on Nikon Ti-E inverted microscope equipped with a Plan Apochromat Lambda 100×/1.49 (magnification/numerical aperture). 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 15 mM Tris(2-carboxyethyl)phosphine hydrochloride (Sigma) for 10 min 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 100 μl 0.2% RapiGest SF surfactant (Waters) and boiled for 10 min and cooled on ice for 10 min. An equal volume of 8 M urea in 0.1 M ammonium bicarbonate (Sigma) solution was added to the samples. BCA (Pierce) was then performed on the samples to estimate protein concentration, and 50 μg of protein was taken for digestion. Blood from EDTA tubes was processed by centrifugation for 10 min at 500g at room temperature to obtain the buffy coat. The supernatant was taken into fresh tubes and spun for 10 min at 2000g to remove platelets. 100 μl of plasma was diluted 1:10 by adding 900 μl of 8 M urea and 0.1 M ammonium bicarbonate solution and stored at −20 °C. 10 μl of the diluted plasma was digested. Proteins were reduced with 5 mM Tris(2-carboxyethyl)phosphine hydrochloride, pH 7.0 for 45 min at 37 °C, and alkylated with 25 mM iodoacetamide (Sigma) 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 9 h at 37 °C. Digestion was stopped using 5% trifluoracetic acid (Sigma) to pH 2 to 3. The peptides were cleaned up by C18 reversed-phase spin columns as per the manufacturer's instructions (Silica C18 300 Å Columns; Harvard Apparatus). Solvents were removed using a vacuum concentrator (Genevac, miVac) and were resuspended in 50 μl HPLC-water (Fisher Chemical) with 2% acetonitrile and 0.2% formic acid (Sigma). Samples were spiked with indexed retention time peptides (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-HPLC system (Thermo Fisher Scientific). Peptides were trapped on precolumn (PepMap100 C18 3 μm; 75 μm × 2 cm; Thermo Fisher Scientific) and separated on an
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