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.