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. 2020 Sep 3;136(10):1169-1179.
doi: 10.1182/blood.2020007008.

Neutrophil extracellular traps contribute to immunothrombosis in COVID-19 acute respiratory distress syndrome

Elizabeth A Middleton  1   2 , Xue-Yan He  3 , Frederik Denorme  1 , Robert A Campbell  1   2 , David Ng  3 , Steven P Salvatore  4   5 , Maria Mostyka  4 , Amelia Baxter-Stoltzfus  4 , Alain C Borczuk  4   5 , Massimo Loda  4   5 , Mark J Cody  1   6 , Bhanu Kanth Manne  1 , Irina Portier  1 , Estelle S Harris  2 , Aaron C Petrey  1   7 , Ellen J Beswick  2 , Aleah F Caulin  8 , Anthony Iovino  6   8 , Lisa M Abegglen  6   8 , Andrew S Weyrich  1   2 , Matthew T Rondina  1   2   9   10 , Mikala Egeblad  3 , Joshua D Schiffman  1   6   8 , Christian Con Yost  1   6
Affiliations

Neutrophil extracellular traps contribute to immunothrombosis in COVID-19 acute respiratory distress syndrome

Elizabeth A Middleton et al. Blood. .

Abstract

COVID-19 affects millions of patients worldwide, with clinical presentation ranging from isolated thrombosis to acute respiratory distress syndrome (ARDS) requiring ventilator support. Neutrophil extracellular traps (NETs) originate from decondensed chromatin released to immobilize pathogens, and they can trigger immunothrombosis. We studied the connection between NETs and COVID-19 severity and progression. We conducted a prospective cohort study of COVID-19 patients (n = 33) and age- and sex-matched controls (n = 17). We measured plasma myeloperoxidase (MPO)-DNA complexes (NETs), platelet factor 4, RANTES, and selected cytokines. Three COVID-19 lung autopsies were examined for NETs and platelet involvement. We assessed NET formation ex vivo in COVID-19 neutrophils and in healthy neutrophils incubated with COVID-19 plasma. We also tested the ability of neonatal NET-inhibitory factor (nNIF) to block NET formation induced by COVID-19 plasma. Plasma MPO-DNA complexes increased in COVID-19, with intubation (P < .0001) and death (P < .0005) as outcome. Illness severity correlated directly with plasma MPO-DNA complexes (P = .0360), whereas Pao2/fraction of inspired oxygen correlated inversely (P = .0340). Soluble and cellular factors triggering NETs were significantly increased in COVID-19, and pulmonary autopsies confirmed NET-containing microthrombi with neutrophil-platelet infiltration. Finally, COVID-19 neutrophils ex vivo displayed excessive NETs at baseline, and COVID-19 plasma triggered NET formation, which was blocked by nNIF. Thus, NETs triggering immunothrombosis may, in part, explain the prothrombotic clinical presentations in COVID-19, and NETs may represent targets for therapeutic intervention.

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Conflict of interest statement

Conflict-of-interest disclosure: C.C.Y. has received grant funding from PEEL Therapeutics, Inc. during the conduct of this study. In addition, C.C.Y. authored a United States patent (patent no. 10 232023 B2) held by the University of Utah for the use of NET-inhibitory peptides for the “treatment of and prophylaxis against inflammatory disorders,” for which PEEL Therapeutics, Inc. holds the exclusive license. A.I. and L.M.A. are consultants and stock option holders of PEEL Therapeutics, Inc., and A.F.C. and J.D.S. are employees and stock option holders of PEEL Therapeutics, Inc. The remaining authors declare no competing financial interests.

Figures

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Graphical abstract
Figure 1.
Figure 1.
Increased plasma NETs correlate with increased COVID-19 severity. (A) We stained for neutrophils and NETs, using immunofluorescence, in lung tissue obtained at autopsy from COVID-19 patients (n = 3). Commercially available normal lung tissue was stained as a negative control. Neutrophils express MPO (red), and early-stage NET-forming neutrophils also express citrullinated histone H3 (green). DAPI serves as a nuclear DNA counterstain (blue). Cyan fluorescence represents the colocalization of citrullinated histone H3 with DNA. The yellow arrowheads point to neutrophils not making NETs, and the white arrows point to neutrophils making NETs. The dashed line highlights a thrombus in the microvasculature (top right panel). Scale bar, 100 μm. Higher-magnification images of the boxed areas in the top row (middle row) are shown along with single-channel images (bottom row); scale bars, 50 μm. (B-F) We used MPO-DNA ELISA to assess NETs in plasma and tracheal aspirate samples from patients in our COVID-19 prospective cohort and in age- and sex-matched healthy donors; each colored dot represents an individual participant. (B) We compared plasma NET levels across all groups: healthy adult donors (n = 17), adults hospitalized with COVID-19 but not intubated for ARDS (n = 22), adults intubated for COVID-19 ARDS (n = 6), and adults recovered from COVID-19 (n = 5). The y-axis depicts plasma NETs expressed as a percentage of healthy adult controls ± SD, arbitrarily set at 100%. (C) We compared plasma NET levels in 2 groups of COVID-19 hospitalized patients: survivors (n = 24) vs nonsurvivors (n = 4). The y-axis depicts plasma NETs expressed as a percentage of healthy adult donors ± SD, arbitrarily set at 100% (dashed line). (D) We correlated plasma NET levels with the Pao2/FiO2 ratio measure of respiratory failure for all hospitalized COVID-19 patients (n = 28). (E) We correlated plasma NET levels with the SOFA Illness Severity Scores for all hospitalized COVID-19 patients (n = 28). (F) We compared plasma NET levels in adult COVID-19 patients (n = 28) with NET levels quantified in the available tracheal aspirate samples of intubated COVID-19 patients (n = 3). The y-axis depicts NET levels expressed as a percentage of healthy adult donors ± SD, arbitrarily set at 100% (dashed line). One-way ANOVA with Tukey’s multiple-comparisons post hoc testing (B-C), Spearman’s rank-correlation test (D-E), Student t test (F).
Figure 2.
Figure 2.
COVID-19 PMNs demonstrate increased activation and NETs at baseline and fail to respond to PMA with increased NET formation. (A) We assessed granularity using flow cytometry in PMNs isolated from healthy donors (n = 6) and COVID-19 patients (n = 4) as a measure of baseline PMN activation. (B-C) We assessed NETs in PMNs isolated from healthy donors (n = 5) and COVID-19 patients (n = 4), with and without PMA stimulation (20 nM; 2 hours), using confocal microscopy and cell-free DNA quantitation. (B) Representative images show NETs (magenta; white arrows) and nuclear DNA (green). Scale bars, 20 μm. (C) We quantified NETs using a fluorescence-based cell-free DNA assay as described in Patients and methods. The y-axis depicts NETs shown as relative cell-free DNA fluorescence ± SEM. Student t test (A), 1-way ANOVA with Tukey’s multiple-comparisons post hoc test (C). n.s., not significant.
Figure 3.
Figure 3.
NETs associate with microthrombi formation and platelet deposition in COVID-19 patients. (A) We stained for NETs and platelets using immunofluorescence in COVID-19 lung tissue obtained from autopsy cases #1 and #3. Early-stage NET-forming neutrophils (MPO, white) express citrullinated histone H3 (green). Microthrombi stained for early-stage NET-forming neutrophils and platelets (PF4; red). DAPI served as a nuclear DNA counterstain (blue). Scale bars, 100 μm (low-magnification, 2 leftmost images). For case #3, higher-magnification images are presented along with single-channel images. Scale bars, 20 μm. Yellow arrows point to thrombi with early-stage NET-forming neutrophils. Colocalized PF4 and early stage NET-forming neutrophils were not present in the analyzed lung sample from case #2. (B) We used flow cytometry to quantify platelet-neutrophil aggregates in whole blood from COVID-19 patients (n = 5) and healthy donors (n = 6). We used ELISA to quantify plasma levels of D-dimer (C), VWF antigen (D), PF4 (E), and RANTES (F) in available plasma from 18 to 22 COVID-19 patients and 5 to 7 healthy donors. Student t test (B), Mann-Whitney or Student t test, depending on the normality of distribution (C-F).
Figure 4.
Figure 4.
nNIF blocks NETs induced by soluble factors in plasma from COVID-19 patients. (A) We used confocal microscopy to assess NET formation qualitatively. We isolated PMNs from healthy adults (n = 4) and used those for experiments testing 16 different available COVID-19 patient plasma samples for NET inductive capacity. We used plasma from age- and sex-matched healthy donors as a control (n = 10). We incubated PMNs with control plasma or COVID-19 patient plasma and assessed for NET formation after a 2-hour incubation. PMNs were preincubated for 1 hour with nNIF (1 nM) or its inactive scrambled peptide control (SCR; 1 nM). Representative images from each experimental arm show NETs (magenta; white arrows) and nuclear DNA (green). Scale bar, 20 μm. (B) We used a high throughput cell-free DNA fluorescence assay to quantify NET formation in the PMNs treated in (A). The y-axis depicts NETs measured as fold change over baseline relative fluorescence units ± SEM. The PMNs treated with control healthy donor plasma serve as the baseline PMN NET level, arbitrarily set at 1. The P values were determined using 1-way ANOVA with Tukey’s multiple-comparisons post hoc testing.
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