RESULTS
NE, MPO-DNA, histone-DNA are dramatically increased and DNase activity was dramatically decreased in COVID-19 ambulatory and hospitalized patients. The blood level of NE, MPO-DNA, histone-DNA and dsDNA of ambulatory and hospitalized COVID-19 patients were dramatically higher than those reported in control subjects. In contrast, the global DNase activity was 10-fold lower in COVID-19 cases. (Figure 1 ). The serum concentration of NE and NET components were not associated with age and sex among groups. The 34 ambulatory cases had a mean age (+/-SD) of 42+/-17 years and a sex ratio (M/F) at 0.88. All had at least two symptoms among fever, dry cough, and dyspnea for less than one week. None had a severe form at this step of the disease. The clinical characteristics and biological findings of hospitalized COVID-19 patients are reported in the Supplementary Tables S1and S2 and those of the 35 healthy controls inSupplementary Table S3 . The concentration of NE was significantly higher in hospitalized vs. ambulatory cases. We observed a higher concentration of NE in patients treated in intensive care vs . local hospitals and medical departments of university hospitals (Figure 1 ). We also observed normal serum concentrations of α1 antitrypsin (AAT), the main blood inhibitor of NE, in hospitalized cases (Supplementary Table S2 ). NE concentrations were distributed in two clusters in ambulatory cases and patients hospitalized in medical department of regional university hospitals. The clusters with the highest concentrations had similar values than those reported in intensive care units (Figure 1 ). Similarly, the concentration of dsDNA was higher in patients admitted in intensive care units (Figure 1 ). We observed highly significant associations of NE with histone-DNA and MPO-DNA (Figure 2 ), while the concentration of dsDNA was associated only with histone-DNA (P=0.03). We also found significant associations of NE with interleukin-6 (IL-6), IL-8 and CXCR2, but not with TNFα (Figure 3 ).
NE, histone-DNA and total DNase activity are associated with severity and multi-visceral manifestations of COVID-19. We reported significant associations of NE with SaO2 at hospital admission, leukocytes, neutrophils, neutrophil to lymphocyte ratio, LDH, markers of cardiovascular and thrombotic risk, including Troponin-T (cTnT), fibrinogen and D-dimer, and markers of renal failure, including urea and creatinine (Table 1 and Figure 3) . Significant association of histone-DNA was reported with the computed tomography score (CT score) of lung damage 13, markers of cardiovascular and thrombotic risk,13 including cTnT and fibrinogen, markers of renal failure, including urea and creatinine and markers of inflammation, including C-reactive protein, ferritin and body temperature (Table 1 and Supplementary Figure S1 ). In contrast, MPO-DNA was associated only with leukocytes (P=0.005) and neutrophils (P=0.004), and at weaker significance, with D-Dimer (P=0.045) and fibrinogen (P=0.047). Total DNase activity was associated with alkaline phosphatase (PAL) and total bilirubin while dsDNA was associated only with ferritin (Table 1 ).
Diagnostic accuracy of NE, MPO-DNA, histone-DNA, dsDNA, DNase activity and related cytokines for detecting disease outcomes in receiver operating characteristic (ROC) analyses . We performed ROC analyses to assess the diagnostic accuracy of NE, MPO-DNA, and histone-DNA for the prediction of disease-related outcomes (Supplementary Table S4 and Figure 4 ). NE had significant thresholds for admission into intensive care units, occurrence of respiratory failure, blood oxygen saturation <85%, and the presence of at least two affected organs (Figure 4 ). The latter was best predicted with a cut-off at 593 ng/mL of NE. Histone-DNA had significant thresholds for admission into intensive care units, kidney injury, and blood oxygen saturation <85%. MPO-DNA had significant thresholds for heart decompensation, kidney injury, respiratory failure, and blood oxygen saturation <85%. The ROC analyses identified also very significant cut-offs for IL-6, IL-8, and CXCR2 for admission into intensive care units, heart decompensation, kidney injury, and respiratory failure. The same observation was made for CXCR2.
Associations between NE >593 ng/mL and patients’ characteristics and outcomes. In univariate analyses, NE >593 ng/mL was significantly associated with CT score, blood oxygen saturation, respiratory failure, presence of at least two affected organs and admission into the intensive care unit (Supplementary Table S5 ). We retained chronic kidney disease, diabetes, and hypertension as potential confounders in multivariable analyses (Supplementary Table S6 ). In the multivariable models that were used to account for the collinearity issue, NE >593 ng/mL was independently associated with CT score and presence of at least two affected organs (Supplementary Table S7 ). The optimal multivariable model had an area under the receiver operating characteristics (AUROC) of 0.876 (95% CI, 0.758–0.950) and a percentage of cases correctly classified of 85%. We performed concordance analyses between cut-offs of NE and cytokines in the prediction of least two affected organs. We reported a 70.4% and 72.7% concordance of NE with CXCR2 and IL-6, respectively (Figure 2 ).
Patient sera decrease the retention of NETs and increase the release of dsDNA in isolated control neutrophils . We studied cell-bound NETs of control neutrophils incubated with patient sera using flow cytometry. We observed a decreased retention of cell-bound NETs, which reflected an increased release, from cells incubated with patientvs. control sera. We further studied the release of dsDNA from control neutrophils produced by patient sera. We observed an increased release of dsDNA from neutrophils incubated with patient vs.control sera (Figure 5 ). The inhibition of NE and other serum serine proteases with 10 µM aprotinin had a weak inhibition effect on dsDNA release. Taken together, these data suggested that the sera from patients increased the release of components of NETs, with a limited role of NE.