FIGURE LEGENDS
Figure 1. Neutrophil elastase (NE), myeloperoxidase – DNA (MPO-DNA), histone-DNA and (b) cell-free dsDNA and total DNase activity in serum of COVID-19 positive cases. Symptomatic ambulatory cases were recruited in screening centers and hospitalized patients in local hospitals and in medical departments and intensive care units of two regional university hospitals. All values were expressed in interquartile range (IQR, 25–75th percentile) NE values: healthy controls 29.5 (23-55), ambulatory 422 (264-659), proximity hospital 405 (224-953), medical department regional hospital 775 (409-1424) and intensive care regional hospital 1366 (1122-1505), the P values between groups were: healthy vs ambulatory <0.0001; healthy vs proximity hospital <0.0001; healthy vs medical department <0.0001; healthy vs intensive care <0.0001; ambulatory vs proximity hospital 0.7870; ambulatory vs medical department 0.0028. ambulatory vs intensive care <0.0001; proximity hospital vs medical department 0.0003; proximity hospital vs intensive care <0.0001 and medical department vs intensive care 0.0239. MPO-DNA values: healthy controls 0.36 (0.23-0.45), ambulatory 1.30 (1.14-1.45); proximity hospital 1.38 (1.28-1.46), medical department regional hospital 1.32 (1.17-1.46), intensive care regional hospital 1.37 (1.32-1.48) and intensive care 0.13 (0.10-0.18), the P values between groups were healthy vs ambulatory <0.0001; healthy vs proximity hospital <0.0001; healthy vs medical department <0.0001; healthy vs intensive care <0.0001; ambulatory vs proximity hospital 0.1237; ambulatory vs medical department 0.8589. ambulatory vs intensive care 0.1276; proximity hospital vs medical department 0.1463; proximity hospital vs intensive care 0.5811 and medical department vs intensive care 0.1482. Histone-DNA values: healthy controls 0.13 (0.10-0.17), ambulatory 0.91 (0.77-1.10), proximity hospital 1.19 (0.91-1.39), medical department regional hospital 1.15 (0.76-1.32) and intensive care regional hospital 1.40 (1.35-1.53), the P values between groups were healthy vs ambulatory <0.0001; healthy vs proximity hospital <0.0001; healthy vs medical department <0.0001; healthy vs intensive care <0.0001; ambulatory vs proximity hospital 0.0013; ambulatory vs medical department 0.0525. ambulatory vs intensive care <0.0001; proximity hospital vs medical department 0.2740; proximity hospital vs intensive care 0.0.0033 and medical department vs intensive care 0.0003. Cell-free dsDNA values: healthy controls 103 (76-138), proximity hospital 120 (90-178), medical department regional hospital 122 (94-207) and intensive care regional hospital 200 (124-286). DNAse activity values: healthy controls 29.25 (22.68-31.48), medical department regional hospital 2.24 (1.66-2.71) and intensive care regional hospital (2.06 (1.61-2.93). The thresholds (dotted lines) were evaluated in healthy controls recruited several months before the epidemy. The threshold of NE was estimated at 73 ng/mL and those of MPO-DNA at 0.562 AU (450 nm absorbance units), histone-DNA at 0.591 AU, total activity of DNase at 9.94 U/mL and serum dsDNA at 95.60 ng/mLc. NE, MPO-DNA and histone-DNA according to the number of organs affected by COVID-19. Data from a , b and c were compared by the Mann-Whitney test.
Figure 2. Associations of neutrophil elastase (NE) with components of neutrophil extracellular traps (NETs), clinical features and biomarkers of severity and multi-visceral harm of COVID . Significant associations of NE were reported with blood concentration of myeloperoxidase (MPO)-DNA and histone-DNA, blood counts of neutrophils, neutrophil/lymphocyte ratio and blood biomarkers of multi-visceral harm, including urea, SaO2, troponin-T, lactate dehydrogenase (LDH), D-dimer, fibrinogen. Correlations were assessed by Spearman rank correlation.
Figure 3. Associations of neutrophil elastase (NE) with cytokines involved in COVID-19 pathological mechanisms . Significant associations of NE were reported with interleukin-6 (IL-6), IL-8 and neutrophil chemokine receptor CXCR2, but not with TNFα. Dotted lines represent the cut-offs reported for multi-organ damage in receiver operating characteristic (ROC) analyses. These cut-offs were used for the concordance analyses. Correlations were assessed by Spearman rank correlation.
Figure 4.  Forest plot reporting the summary of the receiver operating characteristic (ROC) analyses to assess the diagnostic accuracy of neutrophil elastase (NE), myeloperoxidase (MPO)-conjugated DNA, histone-DNA, cell-free dsDNA and DNase activity for the prediction of disease-related outcomes.  For each biomarker, a ROC analysis was performed using the following classification variables: intensive care unit admission, heart decompensation, liver injury, kidney injury, respiratory failure, blood oxygen saturation <85%, number of affected organs ≥2, and NEWS 2 score ≥5. For each ROC analysis, the summary results were reported using the area under the ROC curve, the 95% confidence interval, and the associated P -value (Supplementary Table 3).
Figure 5. Neutrophil innate immunity is a target of the systemic effects of COVID-19. a, b. NET formation and release of Neutrophil dsDNA by sera of COVID-19 patients. a . Representative data of flow cytometry experiments showing NET detection as citrullinated H3 (H3cit) and myeloperoxidase (MPO) double-positive neutrophils (top) and NE-positive neutrophils (bottom), after incubation of neutrophils with sera. b . Quantitative analysis of NET retention on neutrophils from a healthy donor incubated with control or patient sera. c . Quantification of DNA release from neutrophils of a healthy donor stimulated with PMA to induce NET formation and subsequently incubated with 10% serum from controls or patients in the absence or in the presence of aprotinin. The amount of released DNA was considered as 100% in unstimulated neutrophils from the donor.