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.