MATERIAL AND METHODS
Recruitment of patients and clinical characteristics of the
study population. The NICO study (Neutrophil innate Immunity in
COvid-19) was registered by the Institutional Review Board of Clinical
Research (DRCI) of the University Hospital of Nancy (N° 2020PI087) and
approved by the Ethical committee. We performed a cross-sectional
consecutive study of 155 positive patients recruited after informed
consent in a screening center, local hospitals and departments of
medicine and intensive care units of two university hospital centers in
the highest pandemic period of COVID-19 in France. We followed the
EQUATOR and BRISQ guidelines for the reporting clinical and biological
data and the use of biological specimens. The infection was
characterized by RT-PCR on nasal swab specimens. The 34 consecutive
ambulatory subjects attended the screening center in the first week of
the disease, as previously described. 10 We recruited
the 121 patients hospitalized for COVID-19 in medical departments of
local hospitals of the region of Tours, Regional University Hospitals of
Marseille and Nancy. Among them, 13 were treated in intensive care
units. We reported the clinical characteristics and outcomes of
hospitalized patients listed in Supplementary Table 1 . The
scoring of lung damage was evaluated by computed tomography in 5 grades
(CT score), as described 13.
Blood sampling and biological assessment. The sera were used
after completion of biochemical testing ordered by the clinician. The
remaining samples were stored in the same conditions among groups, at
20°C in the 24 hours following blood withdrawal. Routine biochemical
markers were assayed in Cobas 8000 analyzers (Roche Diagnostics,
Switzerland) and Atellica (Eschborn, Germany).
Measurement of elastase, MPO-DNA, DNA-histone complexes, dsDNA
and cytokines in serum. Circulating levels of histone-DNA complexes
were measured in serum by the Cell Death Detection ELISA kit (Roche
Diagnostics, Sigma Aldrich, USA), as described
previously14. Values were reported in 450 nm
absorbance units. DNA-MPO complexes were measured by replacing the
anti-histone antibody with a rabbit polyclonal antibody against
myeloperoxidase (Merck, Darmstadt, Germany). NE concentration was
determined using the Neutrophil Elastase Human ELISA Kit (Invitrogen).
Values were reported in ng/mL. Cell-free dsDNA was quantified using the
QuantiT PicoGreen® dsDNA Assay Kit, after subtraction of background
(Invitrogen). IL-6, IL-8 and CXCR2 were assayed by ELISA kits from
Reagentbio Ireland LTD, Dublin, Ireland, and TNFα by ELISA kit from R&D
Systems Inc.USA, Minneapolis, Minnesota, USA.
Total Deoxyribonuclease (DNase) activity in serum by single
radial enzyme diffusion (SRED) assay. DNase activity was quantified on
agarose gel containing 0.13 mg.mL-1 DNA from salmon
sperm in a buffer containing 100 mM MES pH 6.5, 20 mM
MgCl2, 2 mM CaCl2 and SYBR Safe DNA gel
stain (Invitrogen, Life Technology) as described.52,53Two microliters of serum were located into wells. Gels were incubated
for 24 hours at 37 °C in a humid chamber. The DNA-SYBR fluorescence was
recorded with a fluorescence scanner.
Quantification of cell-bound NETs in neutrophils incubated with
patient serum. To determine whether serum of COVID-19 patients
influence cell-bound NETs, blood from one healthy donor was mixed (1:1
vol/vol) with patient sera prior to PMA treatment and incubated for 6
hours. Red cell lysis was achieved by the addition of 1 mL of FACS lyse
reagent (BD Biosciences; 1:10 dilution) for 5 min. Bovine serum albumin
(BSA, 1 mL at 2%) in phosphate-buffered saline (PBS) was then added and
the mixture was centrifuged at 1000 g for 10 min. The pellet
resuspended in PBS was analyzed using a Gallios flow cytometer (Beckman
Coulter, Villepinte, France). The gating strategy sequentially focused
on (i) CD66 positive cells and then (ii) H3Cit-positive and MPO-positive
cells.
Assay for in vitro NET DNA release of
Neutrophils by patient’s serum. Isolated neutrophils were resuspended
in RPMI 1640 and 1 × 105 cells were seeded in 96-well
tissue culture plates. Plates were incubated at 37°C to allow cells to
adhere. Following stimulation with 100 µL of 0.1 ng/mL PMA for 4h, wells
were incubated with 1 U/mL MNase or DNase1 (both from Worthington) for
10 min or 10 µL patient or control serum for 6 h, and then 2 mM EDTA was
added to stop nuclease activity. The DNA content released in the
supernatant was quantified using the QuantiT PicoGreen® dsDNA Assay Kit
(Invitrogen). The amount of released DNA was considered as 100% in
unstimulated neutrophils of healthy donors.
Statistical analyses. All quantitative variables are shown as
the median and interquartile range (IQR, 25–75th percentile) and
qualitative variables as percentages and 95% confidence interval (95%
CI). Non-parametric tests were used when data distribution was not
normal. Univariate analyses were performed using the chi-squared test or
Fisher’s exact test for categorical variables and the Mann-Whitney U and
Kruskal-Wallis tests and Spearman’s rank correlation for continuous
variables. Post-hoc pairwise comparisons between subgroups were
performed using the Conover test. We subsequently performed receiver
operating characteristic (ROC) analysis to look for the optimal
thresholds associated with disease outcomes 15.
Clinical and biological criteria were used to define organ injuries (see
the Methods section in this article’s Online Repository atwww.jacionline.org).
Bias-corrected and accelerated (BCa)-bootstrap interval after 10,000
iterations for the Youden index and its associated values were performed16. In step #2, using multivariable logistic
regression analysis, we assessed whether the ROC-defined threshold
defined for elastase (>593 ng/L) was associated with a
‘number of affected organs ≥2’ after accounting for potential
confounders. We assessed model discrimination using ROC analysis (AUROC
and 95% CI) and model calibration using the Hosmer and Lemeshow
goodness-of-fit test and Nagelkerke R2 statistics 17.
In step #3, we assessed the association between elastase level
(> or ≤593 ng/L) and patients’ demographics, medical
histories, and disease outcomes. Statistical analyses were performed
using MedCalc, version 19.1 (MedCalc Software, Ostend, Belgium) and
Stata SE, version 12.1 (College Station, Texas, US), based on a
two-sided type I error with an alpha level of 0.05.