key: cord-0983462-z0u7elvp authors: Ouwendijk, Werner J D; Raadsen, Matthijs P; van Kampen, Jeroen J A; Verdijk, Robert M; von der Thusen, Jan H; Guo, Lihui; Hoek, Rogier A S; van den Akker, Johannes P C; Endeman, Henrik; Langerak, Thomas; Molenkamp, Richard; Gommers, Diederik; Koopmans, Marion P G; van Gorp, Eric C M; Verjans, Georges M G M; Haagmans, Bart L title: Neutrophil extracellular traps persist at high levels in the lower respiratory tract of critically ill COVID-19 patients date: 2021-01-27 journal: J Infect Dis DOI: 10.1093/infdis/jiab053 sha: 024003555def81fecbe909a84e8fe5b14f9d3703 doc_id: 983462 cord_uid: z0u7elvp SARS-CoV-2 induced lower respiratory tract (LRT) disease can deteriorate to acute respiratory distress syndrome (ARDS). Because the release of neutrophil extracellular traps (NETs) is implicated in ARDS pathogenesis, we investigated the presence of NETs and correlates of pathogenesis in blood and LRT samples of critically ill COVID-19 patients. Plasma NET levels peaked early after ICU admission and correlated with SARS-CoV-2 RNA load in sputum and levels of neutrophil-recruiting chemokines and inflammatory markers in plasma. Baseline plasma NET quantity correlated with disease severity, but was not associated with soluble markers of thrombosis nor with development of thrombosis. High NET levels were present in LRT samples and persisted during the course of COVID-19, consistent with the detection of NETs in bronchi and alveolar spaces in lung tissue from fatal COVID-19 patients. Thus, NETs are produced and retained in the LRT of critical COVID-19 patients and could contribute to SARS-CoV-2-induced ARDS pathology. A c c e p t e d M a n u s c r i p t BACKGROUND Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been identified as the causative agent of a global outbreak of respiratory tract disease (COVID-19) [1] . As of July 15 th 2020, over 13.3 million COVID-19 cases and more than 578 thousand deaths were reported worldwide [2] . COVID-19 is characterized by range of symptoms including fever, cough, dyspnea and myalgia, but in some patients the infection results in viral pneumonia [3, 4] . About 18% of hospitalized patients develop acute respiratory distress syndrome (ARDS), requiring admission to the intensive care unit (ICU) and mechanical ventilation for a period of several weeks [3, 4] . Pulmonary (hyper)inflammatory responses and coagulopathy are major determinants of disease severity and death in COVID-19 patients [5, 6] . Neutrophils are key players in the pathogenesis of ARDS and have been found to extensively infiltrate lung tissue of COVID-19 patients as well [7, 8] . In addition to killing pathogens through oxidative burst and phagocytosis, neutrophils can produce neutrophil extracellular traps (NETs)web-like structures of DNA, histones, antimicrobial proteins and oxidant enzymes [9] . Excessive NET formation is associated with ARDS triggered by a variety of viruses and bacteria [10] [11] [12] , as well as hypercoagulability [13] . Recent studies demonstrated increased NET levels in plasma of hospitalized COVID-19 patients, as well as the presence of platelet-NET aggregates in plasma and affected lung tissue [14, 15] . Interestingly, plasma NET abundance appeared to correlate with disease severity in hospitalized COVID-19 patients [14, 15] . These results need to be confirmed in larger cohorts and compared to critically ill SARS-CoV-2 infected patients with ARDS. The aim of this prospective cohort study was to investigate the presence of NETs and correlates of pathogenesis SARS-CoV-2 RT-qPCR. SARS-CoV-2 E gene copies per ml sample were determined by reverse-transcriptase linked quantitative PCR (RT-qPCR) as described [16] . His-DNA and MPO-DNA ELISA. Cell-free histone-DNA (his-DNA) complexes were analyzed using the Human Cell Death Detection ELISA PLUS (Sigma Aldrich), using 3,3',5,5'-Tetramethylbenzidine (TMB) as a substrate and measured at 450 nm, using 620 nm as reference. Myeloperoxidase (MPO)-DNA ELISA was performed as described [17] . In brief, wells were coated with mouse anti-MPO antibody (5 µg/ml; clone 4A4, Bio-Rad) overnight at 4˚C. Samples (40 µl) were mixed with incubation buffer and peroxidase-labelled anti-DNA antibody (80 µl/sample; both from the Human Cell Death Detection ELISA PLUS kit; Sigma Aldrich), incubated for 2 hours at room temperature and analyzed using TMB as a substrate. All samples presented in a single figure were measured on the same plate in the same assay. Immunohistochemistry. Post-mortem lung tissues were obtained from 6 COVID-19 patients (Supplementary Table 1 (Table 1 and Supplementary Table 1 ). Patients were between 25 and 77 years old (mean: 63 ± 11.7 years), predominantly male (78%) and mostly had above [13, 19] , and NET-specific MPO-DNA complexes (p<0.001) compared to healthy control subjects ( Figure 1A ). MPO-DNA levels correlated significantly with his-DNA abundance in plasma ( Figure 1B ) and with SARS-CoV-2 RNA load in paired sputum samples of COVID-19 patients ( Figure 1C ). Additionally, MPO-DNA levels correlated weakly with quantities of neutrophilrecruiting chemokines IL-8 and CXCL10 in plasma ( Figure 1D ). Thus, SARS-CoV-2induced ARDS is associated with the presence of NETs in blood. Increased levels of inflammatory markers, including C-reactive protein (CRP), and pro-inflammatory cytokines such as IL-6 are present in blood of COVID-19 patients and are associated with disease severity [3, 4, 6] . We observed that plasma NET levels correlated with CRP and IL-6 levels in patients requiring prolonged ICU admission ( Figure 2D ), but not those released <14 days of ICU admission or with Whereas MPO + H3Cit + neutrophils were found in pulmonary vasculature, bronchi and alveoli, filamentous NETs were mainly observed in alveolar spaces ( Figure 5B ). We observed highly variable numbers of CD61 + platelets, with occasional colocalization of H3Cit + neutrophils and CD61 + platelets ( Figure 6A ). However, the majority of MPO + H3Cit + neutrophils and NETs colocalized with fibrinogen depositions in bronchi and alveoli ( Figure 6B ). Thus, neutrophils infiltrate and undergo NETosis in lung during SARS-CoV-2 induced ARDS. In this prospective cohort study we measured the levels of NETs in the lower respiratory tract and blood of critically ill COVID-19 patients admitted to the ICU, and compared NET abundance to virological, immunological and clinical parameters. Three main findings are reported. First, we showed that NET levels in blood are increased in critical COVID-19 patients, especially early after ICU admission, and correlate with the viral RNA load in sputum and blood levels of neutrophil-recruiting A c c e p t e d M a n u s c r i p t chemokines and inflammatory markers. Second, NET levels were more abundant in lower respiratory tract samples compared to plasma samples of COVID-19 patients and, importantly, were found to persist during ICU admission. Third, we observed that filamentous NETs were mainly present in the alveoli and bronchi, which often contained fibrinogen networks enclosing abundant neutrophils undergoing NETosis. Presence of high quantities of NETs in the lungs contributes to viral and nonviral ARDS [10, 20] . NET components, including extracellular histones and granular proteins like neutrophil elastase and myeloperoxidase, impair the pulmonary epithelial-endothelial barrier by dysregulating epithelial and endothelial cell junctions or inducing cell death, resulting in the accumulation of proteinaceous oedema and inflammatory cells in alveoli [21] [22] [23] . Additionally, NETs induce macrophages to secrete proinflammatory cytokines and stimulate plasmacytoid dendritic cells (pDCs) to release type I IFN [24, 25] , thereby exacerbating the alveolar inflammatory response. Conversely, degradation of NETs, using DNase treatment or pharmacological inhibition of NETosis, prevents damage to the alveolar epithelialendothelial barrier, reduces pulmonary inflammation and improves survival in experimental animal models of bacterial and viral lung disease [10, 26] . Moreover, in murine models of influenza virus-induced ARDS, DNase treatment was shown to improve survival, whereas excessive NET formation was shown to induce ARDS [21, 27] . Our detection of high NET levels in the lower respiratory tract of critically ill COVID-19 patients, combined with the generic mechanisms by which NETs contribute to ARDS, suggest that NETs are likely a pathogenic feature of COVID-19 ARDS. A c c e p t e d M a n u s c r i p t syncytial virus and more recently also SARS-CoV-2, as well as DNA viruses like varicella-zoster virus [12, 15, 20, [28] [29] [30] . Consistent with a role of NETs in exacerbating pulmonary pathology, high quantities of NETs are present in respiratory tract samples from severe COVID-19, influenza pneumonia and varicella pneumonia patients [12, 31] . Interestingly, NET abundance generally correlates poorly between paired plasma and LRT samples of these patients ( Figure 4C ) [12, 31] , possibly related to our observation that most NETs were formed and retained in alveoli and bronchi of COVID-19 patients. Consequently, plasma NET abundance appears to be a better predictor for disease severity in influenza A virus and SARS-CoV-2 infections compared to LRT samples [14, 15, 30, 31] . Coagulopathy is a prominent feature of severe COVID-19 and associated with poor prognosis [5] . NETosis and coagulation are intimately related, with each process capable of promoting the other [23, 32] . Detection of platelet-neutrophil aggregates in blood and microthrombi in affected lung tissue of COVID-19 patients suggest that neutrophils and/or NETs may contribute to SARS-CoV-2 induced coagulopathy [14] . This may involve NET components like extracellular histones, which have been described to cause platelet aggregation [33, 34] . We report that the longitudinal changes of plasma NET levels are not associated with thrombotic events, nor with soluble markers of thrombosis (i.e. fibrinogen, D-dimer and platelet counts); the latter was also observed by Middleton et al. [14] . Nevertheless, pulmonary microthrombi are already present in early stages of some COVID-19 [35] , and may capture and activate primed neutrophils to release NETs [14] . Similar to a recent report [28] we observed that NETs were predominantly located in bronchioles and alveoli. By contrast, the pulmonary vasculature contained many H3Cit + neutrophils [14, 28] , but not NETs, suggesting that neutrophils may be primed by A c c e p t e d M a n u s c r i p t pulmonary microthrombi and only undergo complete NETosis upon release into the fibrinogen-rich alveolar space [36] . Regulated NETosis may contribute to the host immune defence against SARS-CoV-2 infection in asymptomatic or mild cases, e.g. by physically entrapping viral particles and reducing virus replication [20, 37] . Factors contributing to severe COVID-19, including SARS-CoV-2 load in the respiratory tract and levels of proinflammatory cytokines in blood [6, 38] , may concurrently stimulate excessive NET production [11, 13] . Consistent with SARS-CoV-2 induced NETosis, we found that plasma NET levels positively correlated with viral RNA load in sputum samples. Additionally, we observed that NET levels positively correlated with inflammatory markers in blood of critically ill COVID-19 patients, suggesting that circulating cytokines may trigger NETosis. Indeed, serum of COVID-19 patientscontaining high quantities of cytokines [4, 6] was previously shown to induces NETosis in neutrophils of healthy subjects [15] . A limitation of this study, and previous studies [14, 15] , is that only hospitalized patients were analyzed, whereas the majority of SARS-CoV-2 infected individuals develop mild disease [39, 40] . Additionally, the BL and BAL samples analyzed were all obtained from critically ill COVID-19 patients, for diagnostic purposes, at the time of severe lung disease, but not were not available at the convalescent phase of the disease. It will be important to compare plasma NET The authors have no conflicts of interest to declare. 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