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.