key: cord-1003985-b8tmd3v7
authors: Herrera, Victoria L. M.; Walkey, Allan J.; Nguyen, Mai Q.; Gromisch, Christopher M.; Mosaddhegi, Julie Z.; Gromisch, Matthew S.; Jundi, Bakr; Lukassen, Soeren; Carstensen, Saskia; Denis, Ridiane; Belkina, Anna C.; Baron, Rebecca M.; Pinilla-Vera, Mayra; Mueller, Meike; Kimberly, W. Taylor; Goldstein, Joshua N.; Lehmann, Irina; Shih, Angela R.; Eils, Roland; Levy, Bruce D.; Ruiz-Opazo, Nelson
title: A targetable ‘rogue’ neutrophil-subset, [CD11b+DEspR+] immunotype, is associated with severity and mortality in acute respiratory distress syndrome (ARDS) and COVID-19-ARDS
date: 2022-04-04
journal: Sci Rep
DOI: 10.1038/s41598-022-09343-1
sha: de0ffa1314535989735d935aac2126757a9533ab
doc_id: 1003985
cord_uid: b8tmd3v7

Neutrophil-mediated secondary tissue injury underlies acute respiratory distress syndrome (ARDS) and progression to multi-organ-failure (MOF) and death, processes linked to COVID-19-ARDS. This secondary tissue injury arises from dysregulated neutrophils and neutrophil extracellular traps (NETs) intended to kill pathogens, but instead cause cell-injury. Insufficiency of pleiotropic therapeutic approaches delineate the need for inhibitors of dysregulated neutrophil-subset(s) that induce subset-specific apoptosis critical for neutrophil function-shutdown. We hypothesized that neutrophils expressing the pro-survival dual endothelin-1/VEGF-signal peptide receptor, DEspR, are apoptosis-resistant like DEspR+ cancer-cells, hence comprise a consequential pathogenic neutrophil-subset in ARDS and COVID-19-ARDS. Here, we report the significant association of increased peripheral DEspR+CD11b+ neutrophil-counts with severity and mortality in ARDS and COVID-19-ARDS, and intravascular NET-formation, in contrast to DEspR[-] neutrophils. We detect DEspR+ neutrophils and monocytes in lung tissue patients in ARDS and COVID-19-ARDS, and increased neutrophil RNA-levels of DEspR ligands and modulators in COVID-19-ARDS scRNA-seq data-files. Unlike DEspR[-] neutrophils, DEspR+CD11b+ neutrophils exhibit delayed apoptosis, which is blocked by humanized anti-DEspR-IgG4(S228P) antibody, hu6g8, in ex vivo assays. Ex vivo live-cell imaging of Rhesus-derived DEspR+CD11b+ neutrophils showed hu6g8 target-engagement, internalization, and induction of apoptosis. Altogether, data identify DEspR+CD11b+ neutrophils as a targetable ‘rogue’ neutrophil-subset associated with severity and mortality in ARDS and COVID-19-ARDS.

To independently test detection of DEspR+CD11b+ neutrophils in NHV blood samples, collaborators performed flow cytometry analysis on Li-heparin anticoagulated NHV whole blood that would allow more robust neutrophil-activation without EDTA-chelation of Ca 2+ and Mg +2 ions. As expected, testing of Li-heparin anticoagulated NHV whole blood detected higher DEspR+CD11b+ activated neutrophils: ~ 52.4% in 37 °C 1-h incubation in buffer (Fig. 1F) , and ~ 93.5% with LPS-induced TLR-4 activation (Fig. 1G ). To assess potential intracellular stores, collaborators analyzed semi-permeabilized TLR4-activated neutrophils via 2% paraformaldehyde (PFA). Flow cytometry analysis detected fourfold higher fluorescence intensity levels of DEspR+ expression but same % of DEspR+CD11b+ neutrophils in PFA-semi-permeabilized neutrophils (Fig. 1H ), compared to non-PFA-permeabilized TLR4-activated neutrophils (Fig. 1G ). This observation suggests intracellular stores of DEspR, in addition to cell surface DEspR, hence higher "total" DEspR+ expression intensity, concordant with cell membrane, cytoplasmic and nuclear DEspR+ expression detected by immunocytology (Fig. 1A, Supplementary Fig. S1A ).

Relevant to ex vivo analysis, these observations indicate that EDTA-anticoagulated blood exhibit less susceptibility to ex vivo experimental changes with increases in time and temperature (Fig. 1E, Supplementary Fig. S1C ). For quantitative ex vivo ARDS patient sample analysis, we therefore used only EDTA anti-coagulated whole blood processed within 1-h from sampling from hereon, in order to minimize confounders that increase DEspRexpression ex vivo. This will avoid overestimating actual circulating levels in patient samples and false positives.

To further confirm DEspR+ expression in neutrophils, we analyzed the requisite co-expression of DEspR and adenosine deaminase acting on RNA (ADAR1) RNA-editase, since DEspR protein-translation is RNA-editase dependent 18 . Using flow cytometry analysis of PFA-fixed, Triton-X permeabilized human neutrophils, we show that every DEspR+ neutrophil is ADAR1+ (Fig. 1I-K) , as seen in DEspR+ Panc1 tumor cells 18 . Co-expression in neutrophils exhibits 0.51 to 0.74 Pearson correlation coefficient, p < 0.0001 (Fig. 1J,K) , thus supporting ADAR1+ neutrophils as a marker corroborating DEspR+ human neutrophils.

To determine whether DEspR+ neutrophils are present in ARDS patient lung tissue, we performed immunohistochemistry analyses of post-mortem serial lung-tissue sections from patients with ARDS (n = 8) in regions of diffuse alveolar damage ( Fig. 2A -I) as well as, in areas of alveolar-capillary injury (Fig. 2J-K) . Using an anti-DEspR mouse-recombinant mAb of hu6g8, hu6g8-m, immunohistochemistry with DAB chromogen (IHC-DAB) was optimized in order to track spatial patterns of DEspR+ expression in lung tissue-sections. This detected DEspR+ neutrophils in intrabronchiolar exudate, along with some DEspR(−) neutrophils identified by the characteristic polylobulated nuclei (Fig. 2B) .

In adjacent serial sections, IHC-DAB immunostaining conditions limited to detection of only high myeloperoxidase (MPO[high])+ expression typical in neutrophils with 5 × higher expression than monocytes 31, 32 ,detected primarily MPO[high]+ neutrophils in the intrabronchiolar exudate (Fig. 2C ). In areas of diffuse alveolar damage (DAD) ( Fig. 2A ,D-F), IHC-DAB analyses of serial sections detected intra-alveolar, intravascular and intraparenchymal DEspR+ neutrophils and monocytes (Fig. 2E) and MPO[high]+ neutrophils (Fig. 2F) . Similarly, in other lung areas with acute alveolar-capillary injury changes (Fig. 2G) , we also detected DEspR+ neutrophils and monocytes in the intravascular, parenchymal and intra-alveolar spaces (Fig. 2H) . Confirmatory IF-staining detected intra-alveolar and interstitial DEspR+CD11b+ neutrophils and macrophages in ARDS (Fig. 2I , Supplementary Fig. S2A ) and COVID-19-ARDS (Fig. 2J, Supplementary Fig. S2B ) lung tissue sections, as well as DEspR+MPO+ neutrophils and macrophages in COVID-19-ARDS lung tissue section (Fig. 2K , Supplementary  Fig. S2C ). These observations validate measuring peripheral levels of DEspR+CD11b+ neutrophils and monocytes by flow cytometry in ARDS and COVID-19-ARDS patient whole blood samples.

RNA-edited transcript 18 is not discernable in scRNA-sequencing limited to 300 nucleotides from the poly-A sequence of each transcript to ascertain specificity and equivalent representation, we studied the DEspR-path- Comparative scRNA-seq analysis showed that positive modulators of DEspR transcription (TLR4 and hypoxia inducible factor 1-alpha, HIF1A), DEspR RNA-editing for translation (ADAR1 RNA-editase), DEspR cell-surface mobilization (TLR4), TLR4-endogenous activators (alarmins S100A8/S100A9) and DEspR downstream-function marker (Mcl1), are all predominantly expressed in neutrophils, along with DEspR's two ligands, ET1 (EDN1), and the signal peptide in VEGFA-propeptide (vegf-SP) (Fig. 3A) . Interestingly, neutrophil expression of the endothelin converting enzyme (ECE1), required for release of ET1 from its pro-peptide ( Fig. 3A) suggests neutrophil self-sufficiency to produce mature ET1, and hence, a putative ET1/DEspR autocrine loop. In contrast, transcripts for other ET1 receptors: ETA-receptor (ETAR) and ETB-receptor (ETBR), VEGF-A receptor: (KDR), and the other S100A8/A9 alarmins receptor: (AGER) were minimally, if not at all, detected in neutrophil scRNAsequences ( Supplementary Fig. S3A-D) .

Furthermore, expression levels of all 9 genes representing DEspR's expression and functional network are significantly increased in COVID-19 compared to healthy controls (Fig. 3B) , and in neutrophils compared to monocytes-macrophages in nasopharyngeal and broncho-lavage COVID-19 samples (Fig. 3C ). The basis to study neutrophils is further supported by scRNA-seq documentation of increased expression of receptors to cytokines increased in ARDS 33 and/or in COVID-19-ARDS 34 , such as: interleukin (IL)-IL-6, IL-8, IL-1β, IL-18, and tissue necrotic factor-alpha (TNF-α) 35 (Supplementary Fig. S3G ). These observations provide molecular evidence linking neutrophils as effectors of "cytokine storms" in ARDS, concordant with the known association of increased neutrophils with delayed apoptosis and mortality in ARDS 1,36,37 and COVID19-ARDS 38 .

To further study the DEspR+ neutrophil-subset, we completed a prospective pilot observational study of consented patients diagnosed with ARDS based on the Berlin Definition before the COVID19 pandemic. We prospectively studied consented ARDS patients (pre-COVID pandemic) regardless of the underlying acute disease trigger and associated comorbidities (Supplementary Table S1 for demographics) .

First, we ascertained DEspR-specific immunotyping of whole blood samples from ARDS patients by validating our gating strategy for flow cytometry ( Supplementary Fig. S4A-E) , DEspR-specific detection in duplicates (Supplementary Fig. S4F -I) and reproducibility in triplicates (Fig. 4A, Supplementary Fig. S4J ). We note that the level of DEspR+CD11b+ neutrophil counts was not simply due to age in ARDS and COVID-19-ARDS (Supplementary Fig. S4K ,L respectively).

With this ascertainment, we then prospectively studied 19 ARDS patients (pre-COVID-19 pandemic), then 11 COVID-19-ARDS patients in the ICU at Boston Medical Center, by FCM-analysis. To assess for putative ; #2: altered nucleus with DNA-rimming towards cell membrane; #3: decondensing  nucleus and some extrusion; #4 and #5: NET-forming neutrophils with extruded DNA, residual nuclei  and intact cell membrane. Panel: hu6g8-AF568 DEspR+ red circle, DAPI blue circle, merged pink circle; Bar = 20 μm. (See Supplementary Fig. S1A ). (B) Immunofluorescence of a representative extruded neutrophil extracellular trap (NET) exhibiting DAPI+ DNA with MPO+ and DEspR+ components. Bar = 10 μm. (C) Left: bar graph showing semi-quantitation of % DEspR+ neutrophils and NET-forming neutrophils in 6 high power fields per slide in 3 slides prepared from NHV polled neutrophils. Right: Western blot analysis showing DEspR expressed in normal human kidney (HK), neutrophils (HN1) and LPS-stimulated neutrophils (HN2), MW, molecular weight markers. LB, Laemli buffer as negative control. (D) Flow cytometry (FCM) analysis of EDTA-anticoagulated NHV whole blood, non-stimulated. Gating of white blood cells for neutrophils, monocytes, lymphocytes and red blood cells (RBC) by their respective clouds determined by differential forward scatter (FSC, x-axis), side scatter (SSC, Y-axis).features, and fluorescence markers: anti-CD11b-FITC, anti-DEspR (hu6g8)-AF568. (E) FCM analysis of NHV (EDTA-anticoagulated) whole blood stimulated with LPS (75 μg/ml, 1-h at 37 °C), Panels left-to-right: FSC/SSC gating for neutrophils; isotype controls hIgG4-AF568, mIgG2-AF488; FMO-DEspR); CD11b (X-axis) vs DEspR (Y = axis Fig. S4J ). With experimental ascertainment of reproducibility of DEspR-specific immunotyping, from hereon, studies were done in duplicates. In COVID19-ARDS patients, we prospectively studied 11-subjects (Supplementary Table S1 for demographics). Mandated by institutional safety requirements, we studied disinfected (4% paraformaldehyde or PFA) whole blood samples from COVID-19-ARDS patients, and performed FCM analysis within 1 h from blood draw. FCM-analysis of subjects representing extremes of the clinical severity spectrum also detected increased total number DEspR+ neutrophils and monocytes in a patient with severe COVID-19-ARDS requiring 61 days intensive care unit (ICU)-care (Fig. 4D ), compared with a patient with milder COVID-19-ARDS discharged after 6 days in the ICU (Fig. 4E ).

Observing differential levels at the polar ends of the clinical spectrum of severity, we next stratified mortality outcomes in ARDS (Fig. 4F ) and COVID-19-ARDS (Fig. 4G ) patients by levels of DEspR+CD11b+ neutrophilcounts (K/μL whole blood). These pilot study trend-maps show an emerging differential pattern between survivors and non-survivors in ARDS and COVID-19-ARDS, providing bases for correlation analyses.

Association of DEspR+ CD11b+ neutrophil-subset with ARDS severity and mortality. To dissect the differential pattern emerging between survivors and non-survivors (Fig. 4F ,G), we first performed correlation matrix analysis on a panel of DEspR-based flow cytometry markers, clinical markers of ARDS severity, and plasma biomarkers associated with neutrophil-mediated secondary tissue injury, and ET1 one of two DEspR ligands (Fig. 5A , Table 1 ). To assess clinical severity, we studied the number of ICU-free days at day 28 from ARDS diagnosis as a measure of mortality (death scored as [-1]) and speed to recovery within 28-days 39 , ARDS severity (SpO2/FiO2 or S/F ratio measure of hypoxemia), and Sequential Organ Failure Assessment (SOFA) scores on the day of sampling for flow cytometry analysis (t1-SOFA) and on day before ICU-discharge or ICU-death (t2-SOFA). To assess context in ARDS pathogenesis, we studied biomarkers of pathogenic events in ARDS relevant to neutrophil-mediated secondary tissue injury: interleukin-6 (IL-6 marker of cytokine storms), soluble C5b9 (terminal complex of complement activation), myeloperoxidase or MPO (neutrophil activation), ratio of the number of copies of mitochondrial to nuclear DNA in plasma (mtDNA-NET-formation) 24 , and DEspR+CD11b+ cytoplasts, (anuclear remnants of neutrophils associated with "vital NETosis") 25 .

Spearman rank correlation matrix analysis detected significant negative correlation between the number of DEspR+CD11b+ activated neutrophils and ICU-free days at day 28 (Fig. 5A , Table 1 ). Other DEspR-based FCMparameters, such as % of DEspR+CD11b+ neutrophils, monocytes and lymphocytes, also showed significant negative correlation with ICU-free days at day 28 (Table 1 ), in contrast with DEspR[-] neutrophil-counts which exhibited no significant correlation (Table 1) . Spearman correlation analysis also detected significant correlation of number (#) and per cent (%) of DEspR+CD11b+ neutrophil with t2-SOFA scores but not with t1-SOFA (Fig. 5A , Table 1 ), supporting a pathogenic role in MOF-progression. In contrast, DEspR[-] neutrophil-counts, NLR, plasma levels of IL-6, MPO, and sC5b9 did not correlate with any of the measures studied (Fig. 5A , Table 1 ).

Next, we analyzed differences in group medians between ARDS-patient survivors and non-survivors ( Fig. 5B-G) . In this pilot study, we found no significant difference between survivors and non-survivors for clinical measures of ARDS severity: S/F ratio (Fig. 5B ), t1-SOFA scores (Fig. 5C) , and prognostic measure NLR (Fig. 5D ). In contrast, significant difference in medians (Mann Whitney p = 0.0001) with large effect size (Hedge's g > 0.8) was detected for DEspR+CD11b+ neutrophil-counts (#) (Fig. 5E ) and % DEspR+CD11b+ neutrophils www.nature.com/scientificreports/ ( Fig. 5F ), but not for DEspR [-] neutrophil-counts (Fig. 5G ). Kaplan Meier survival curve analysis with a threshold for DEspR+CD11b+ neutrophil-counts set at 3,000/μL (3 K/μL) whole blood showed significant differences in survival (P < 0.0001) (Fig. 5H ).

ilarly, in COVID-19-ARDS pilot group, Spearman rank correlation matrix analysis showed significant, strong, negative correlation of DEspR+CD11b+ neutrophil-counts with ICU-free days at day 28 from ARDS diagnosis, and with ARDS severity S/F ratio, in contrast to no correlation with DEspR[-] neutrophil-counts (Fig. 5I , Table 2 ). Interestingly, the sum of %DEspR+ [monocytes and neutrophils] correlated with ICU-free days at day 28 with higher Spearman correlation coefficient, significance and power than either alone (Table 2 ). This observation is concordant with neutrophil-monocyte intravascular-interactions reported to contribute to systemic tissue injury in acute glomerular injury 40 . In contrast, the neutrophil lymphocyte ratio (NLR) showed significant albeit less robust correlation with ICU-free days at day 28 in COVID-19-ARDs compared to DEspR+CD11b+ neutrophil-counts. Comparative analysis of COVID-19-ARDS survivors and non-survivors showed significant differences in medians for S/F ratio ( Fig. 5J ) but not for t1-SOFA score (Fig. 5K ). Concordant with correlations detected, significant differences and large effect size were also detected between survivors and non-survivors for NLR (Fig. 5L ), DEspR+CD11b+ neutrophil-counts (Fig. 5M ), but not for DEspR[-] neutrophils (Fig. 5N) .

To compare our pilot study observations with emerging biomarkers of severe COVID-19, we performed a retrospective analysis of COVID-19-ARDS patients requiring ventilatory support at Boston Medical Center. Data corroborate significant differences in NLR (Supplementary Fig. S5A -D), concordant with reports that increased NLR is an independent predictor of mortality in ARDS and COVID-19 41 . In contrast, C-reactive protein did not show significant differences between survivors and non-survivors (Supplementary Fig. S5E -H).

NET-forming neutrophils relevant to detection of increased levels of NETs in in severe COVID-19 10, 11, 42, 43 , we performed immunofluorescence staining to directly visualize and quantify NET-forming neutrophils in whole blood smears prepared from COVID-19-ARDS patients within 1-h from blood draw. Using high-resolution confocal imaging of immunofluorescent-staining for DEspR+CD11b+ expression, we detected differential levels of DEspR+CD11b+ NET-forming neutrophils in ARDS non-survivor, compared with ARDS-survivor and ICUpatient non-ARDS survivor (Fig. 6A) .

To further characterize NET-forming neutrophils, we immunostained for MPO-positivity, a hallmark of NETs. IF-staining of blood smears detected MPO+DNA and, interestingly, DEspR+DNA in NET-forming neutrophils with the classical cytolytic NET-formation (suicidal NETosis) phenotype (Fig. 6B ). Both MPO+ or DEspR+ or dual MPO+/DEspR+ micro-vesicles are detected in the DAPI+DNA-cloud and DNA-strands (Fig. 6B ). In the same image, a non-cytolytic NET-forming neutrophil with extruded DNA but no vesicles and a nucleus that is DEspR+MPO+DAPI+ (Fig. 6B) . We observed clustering of NET-forming neutrophils in COVID-19-ARDS non-survivors compared with COVID-19-ARDS survivors (Fig. 6C ). We also observed interconnecting filamentous-DNA networks ( Fig. 6D) with DEspR+ CD11b+ subcellular 'beads' along the DNA-strands in both ARDS and COVID-19-ARDS patient blood smears (Fig. 6E, Supplementary Fig. S6A ,B).

to quantify DEspR+CD11b+ NET-forming neutrophils (Ns), we used shape analysis (Fig. 6F ). Semi-quantitative confocal microscopy distinguished NET-forming neutrophils with low circularity index, from non-NETforming neutrophils with expected high circularity of 1.0 (see Supplementary Methods). Quantitative analyses , and corresponding scRNA-seq levels of DEspR-modulators: HIF1A, modulator of DEspR transcription, at RNA level (HIF1A stabilized in normoxia by activated TLR4), RNA-editing of DEspR transcript (ADAR1 RNA-editase), mobilization to cell surface upon activation (TLR4), DEspR-bioeffect marker for pro-survival role (Mcl1), DEspR-ligands ET1 (EDN1, ECE1), and VEGFAsignal peptide (propeptide VEGF), and TLR4-activators during cell injury -alarmins: S100A8 and S100A9. Yellow open circle, encircles estimate (≈) DEspR+ neutrophils based on flow cytometry findings that every DEspR+ neutrophil is ADAR1+. DEspR-RNA edited mRNA is beyond scRNA-seq 300 nt-seq from poly-A tail. No expression (blue circle), ≥ 2 × expression (filled circle). (B) Quantitative scRNA-seq database analysis of DEspR-expression network genes measured as % of cells in sample with ≥ 2x-fold increase in RNA levels in nasopharyngeal samples comparing non-COVID-19 control patients (n = 5, mean ± sd: 3.1 ± 1.9), mild COVID-19 patients (n = 8: 42.6 ± 10.2), and critically ill COVID-19 patients (n = 11: 54.1 ± 10.6). Non-parametric one-way ANOVA with Tukey's multiple comparisons testing: *, p = 0.0226, ****, p < 0.0001. (C) Quantitative scRNA-seq database analysis of DEspR-expression network genes comparing % ≥ 2 × expression for each gene among neutrophils (Ns) and monocytes-macrophages (Ms) in nasopharyngeal (np) and broncholavage fluid (blf) samples, (n = 9 genes in DEspR-expNetwork per cell type) in COVID-19-patient samples. Average % expression of all 9 genes in Ns-np: (mean ± sd: 46.78% ± 19.9); Ms-np: (12.7% ± 10.7); Ns-blf: (55.3% ± 25.1), and Ms-blf (4.2% ± 2.8). Non-parametric one-way ANOVA with Sidak's multiple comparisons testing: ***, p = 0.0006; ****, p < 0.0001. www.nature.com/scientificreports/ of COVID-19-ARDS patient samples spanning hundreds of neutrophils per slide showed significant strong negative correlation of #DEspR+NET-forming neutrophils with mean circularity index per patient (Spearman rho = 0.78, p = 0.006, power > 0.8) (Fig. 6G ). With this correlation, we used a circularity index < 0.8 to identify NET-forming neutrophils for quantitative analyses.

In COVID-19-ARDS patients, the total number of DEspR+CD11b+ NET-forming neutrophils correlated strongly with three clinical measures: 1] outcome at day-28 (ICU-free days at day 28), 2] degree of hypoxemia (SpO2/FiO2 or S/F ratio), and 3] severity of multi-organ failure score at end of ICU-stay (t2-SOFA), but not with t1-SOFA (Table 2 , Fig. 5I) . Concordantly, analysis of number of DEspR+CD11b+ NET-forming neutrophils detected significant differences (p < 0.05) in medians between COVID-19-ARDS survivors and non-survivors (Fig. 5O ). In contrast, the average or peak D-dimer levels, indicative of thrombosis risk, did not exhibit significant differences between COVID-19-ARDS survivors and non-survivors ( Supplementary Fig. S5I-L) . Additionally, scRNA-seq profile for PADI4 linked to "suicidal NETosis" shows low (1.4%) number of neutrophils expressing PADI4 > 2X fold in COVID-19-ARDS neutrophils ( Supplementary Fig. S3E ).

Having detected DEspR+CD11b+ NET-forming neutrophils and DEspR+ MPO+ NETs on immunostained whole blood smears (Fig. 6A,B) , we analyzed levels of cytoplasts as circulating NET-remnants by flow cytometry. We and another research site detected elevated DEspR+CD11b+ cytoplast levels in ARDS subjects (Fig. 6H ) however, we did not observe association between circulating DEspR+CD11b+ cytoplast levels with clinical measures of ARDS severity (Fig. 5A , Table 1 ). Nevertheless, an independent small pilot study of patients with sepsis, and sepsis-ARDS confirmed elevated DEspR+CD66+ cytoplasts 44 and neutrophils in contrast to low to no levels in healthy donors ( Supplementary Fig. S6C-H) . As a marker for neutrophil-degranulation, CD66b confirms neutrophil-derived cytoplasts. We note that neutrophils were isolated from whole blood samples via inertial microfluidic separation from RBCs 45 , performed however, ~ 3-h from blood draw-hence accounting for the much higher levels > 90% DEspR+ cytoplasts and neutrophils observed.

DEspR-inhibition, we analyzed ARDS patient whole blood with humanized anti-DEspR IgG4 S228P antibody, hu6g8, added as ex vivo treatment for 17-20 h overnight with rotation to prevent aggregation. Controls comprised of patient-specific mock-treated and baseline pre-treatment controls (Fig. 7A) . Comparative FCM-analysis showed that compared to baseline levels and after 17-20 h of ex vivo incubation at 37 °C, DEspR+ neutrophils increased in number compared with markedly decreased number of DEspR[-] neutrophils (Fig. 7A,B) , suggesting that normal neutrophilic constitutive apoptosis is delayed in DEspR+ neutrophils but not in DEspR[−] neutrophils, and that some DEspR[−] neutrophils became DEspR+ with time.

To determine DEspR's role in delayed apoptosis, we inhibited DEspR via hu6g8-treatment, and performed flow cytometry after 17-20 h of treatment ex vivo. This showed that hu6g8 decreased the number of DEspR+ neutrophils in ARDS patient whole blood (Fig. 7B ). Hu6g8 also reduced myeloperoxidase (MPO) (Fig. 7C) and soluble terminal complex of complement (sC5b9) (Fig. 7D ) plasma levels, in contrast to greater than twofold increased levels in mock-treated controls, respectively after 17-20 h ex vivo incubation. These observations indicate that hu6g8 induced neutrophil apoptosis and function-shutdown of neutrophil-complement system reciprocal co-activation after 17-20 h of DEspR-inhibition via hu6g8-treatment. Importantly, neutrophil scRNAseq profile for CD47, the "don't eat me signal" is minimal, with only 0.3-0.91% of neutrophils with > 2 × fold CD47 (n = 19 COVID-19 patients) ( Supplementary Fig. S3F ). This supports the therapeutic hypothesis that induction of apoptosis in DEspR+ neutrophils with no CD47 "don't eat me signal" can be cleared by efferocytosis.

To further test that DEspRinhibition induces apoptosis in DEspR+ CD11b+ neutrophils, we performed live cell imaging of Rhesus macaque neutrophils exposed to fluorescently labeled hu6g8-AF568 or fluorescently labeled human IgG4-AF568 isotype control for 20 min at 4 °C to avoid non-specific endocytosis. We selected Rhesus neutrophils as model system since Rhesus-to-human neutrophils are more similar than human-to-mouse neutrophils 46 .

We first validated the presence of circulating DEspR+ CD11b+ neutrophils in Rhesus via flow cytometry using identical conditions to ex vivo analysis of ARDS patient samples ( Supplementary Fig. S7A-H) . Next, we studied hu6g8 target engagement, internalization, and induction of characteristic apoptosis cell-budding bioeffects by confocal live cell imaging of Rhesus neutrophils. We exposed Rhesus neutrophils to either AF568-labeled anti-DEspR hu6g8 antibody (treatment) or IgG4-isotype (mock-treatment) control for 20 min at 4 °C to avoid non-specific endocytosis. After removing excess unbound antibody, 24-h live cell imaging was initiated with video-recordings. At t-45 min, live-cell images detected target engagement and internalization of hu6g8-AF568 antibody (Fig. 7E, Supplementary Fig. S7I ) but not in the isotype control (Fig. 7F ). Specificities were confirmed throughout with representative t-12 h timepoint images (Fig. 7G,H) . At t-12 h, live cell imaging showed more apoptotic cell budding changes in NHP-neutrophils with internalized hu6g8 (Fig. 7G ). In the isotype control, apoptotic cell budding was detected concordant with neutrophil constitutive apoptosis (Fig. 7H) . SytoxGreen impermeable dye uptake marked loss of cell viability. Both cell death indicators increased with time (Fig. 7G,H) .

At the 12-h midpoint, quantitation of apoptotic cell changes and SytoxGreen-positive non-viability were done. Quantitative analysis of 18 high power fields (HPFs) with 20-50 cells/HPF representing three independent experimental fields of view showed that hu6g8 induced apoptosis in DEspR+ neutrophils significantly greater than levels seen in isotype-treated control NHP cells (Fig. 7I) . Importantly, hu6g8 induced apoptosis greater than constitutive apoptosis occurring in DEspR[-] cells unaffected by hu6g8 treatment (Fig. 7I) . Interestingly, loss-ofviability staining by Sytox Green occurred in neutrophils not undergoing apoptotic cell budding, and was also www.nature.com/scientificreports/ slightly greater in hu6g8-treated neutrophils compared with isotype mock-Tx controls (Fig. 7J) , indicating that DEspR-inhibition may facilitate other programmed cell-death in neutrophils via decreased CIAP2 as observed in anti-DEspR mAb-treated pancreatic cancer stem cells 47 . Interestingly, shortly after the addition of SytoxGreen at t-15 min of live cell imaging, NET-formation with cytolysis was observed with DEspR+nucBlue+SytoxGreen+ NETs (Fig. 7K-M) . DEspR+ neutrophils with internalized AF568-labeled hu6g8 were also observed (Fig. 7K ). This contrasts t-12 h (Fig. 7G) where no NET-formation events were observed up to 24 h, suggesting the hypothesis that anti-DEspR induces neutrophil-apoptosis and pre-empts NET-formation. Lastly, NHP neutrophils shown to be > 90% DEspR+ at baseline flow cytometry analysis and day-1 live cell imaging (Fig. 7N ) exhibited marked delayed apoptosis as live DEspR+ neutrophils were still observed 6-days after blood sampling (Fig. 7O ).

Data from multiple experimental systems among multi-center collaborators testing neutrophils in whole blood samples, either from NHVs, ARDS or COVID-19-ARDS patients, or Rhesus macaques, reproducibly identify the DEspR+ CD11b+ neutrophil-subset, concordant with cumulative evidence for neutrophil heterogeneity 1, 48 . Detection of DEspR+ intracellular stores and increase in DEspR+CD11b+ neutrophils upon LPS-TLR4 activation in NHV blood samples indicate a dynamic subset-response to TLR4-activation. Importantly, the identification of the DEspR+CD11b+ neutrophil-subset in whole blood is supported by detection of DEspR+CD11b+ and DEspR+MPO+ neutrophils and monocyte/macrophages in postmortem lung tissue sections from ARDS and COVID-19-ARDS. Detection in the lung interstitium, intra-alveolar spaces, and vascular lumen in association with either diffuse alveolar damage (DAD) or alveolar-capillary injury confirm identification of the DEspR+ neutrophil-subset.

In marked contrast to DEspR[-] neutrophils, the subset-specific correlations of DEspR+CD11b+ neutrophil-counts with severity and mortality measures in both ARDS and COVID-19-ARDS suggests pathophysiological relevance of DEspR+CD11b+ neutrophils as a neutrophil-subset. Additionally, the characteristics of DEspR+CD11b+ neutrophils observed in ex vivo analyses: delayed apoptosis, a predisposition to intravascular NET-formation and neutrophil-NET clustering-further support DEspR+CD11b+ neutrophils as a , and > 28 days in the ICU = 0; SpO2/FiO2 or S/F ratio, Sequential Organ Failure Assessment (SOFA) scores on day of FCM-analysis (t1-SOFA), SOFA on day of ICU-discharge or ICU-death (t2-SOFA)]; 2] plasma biomarkers reported to be elevated in ARDS by others [interleukin-6 (IL-6), endothelin-1 (ET1), myeloperoxidase (MPO), terminal complex of complement (sC5b9), and ratio of number of copies of mitochondrial/nuclear DNA (mt/nucl DNA), and 3] flow cytometry parameter linked to NET-formation (cytoplast levels with high or low granularity (SSC). See Table 1 for specific values: correlation coefficient rho, P-values with power > 0.8. Graphs of ARDS survivors (S, n = 13), non-survivors (non-S, n = 6): (B) S/F ratio as indicator of hypoxemia, survivors (mean ± sd: 214.7 ± 62.1), non-survivors (164.9 ± 60.8), p value not significant; (C) SOFA score, survivors (mean ± sd: 5.4 ± 3.1) and non-survivors www.nature.com/scientificreports/ pathophysiological 'rogue' neutrophil-subset. Notably, these characteristics of DEspR+CC11b+ neutrophils are concordant with observations of neutrophil changes in ARDS and COVID19-ARDS patients 1, 11, 30, 36 . Consistent with expectations of a 'rogue' neutrophil-subset implicated in progression of ARDS and COVID-19-ARDS, increased RNA levels of the DEspR-pathway gene network detected in neutrophil-specific COVID-19-ARDS scRNA-seq data files interconnect in multiple positive autocrine loops. These autocrine loops span not just ligand-receptor levels, (DEspR, ET1, VEGF-sp), but also transcriptional modulators of DEspR itself, its ligands and required RNA-editase (HIF1A and TLR4), and DEspR's modulator for cell-surface expression (TLR4) and TLR4's endogenous ligands (S100A8/A9). These autocrine loops provide a potential self-sustaining framework that would be expected of a neutrophil-subset implicated in progressive MOF. These observations are concordant with elevated TLR4 49 , S100A8/A9 14,49,50 , ADAR1 51 , and ET1 52 detected in clinical (ARDS, or COVID19-ARDS) and pre-clinical model studies of ARDS, COVID-19-ARDS.

The direct visualization of DEspR+CD11b+ neutrophils with extruded DNA and intact nucleus and cell membrane in ARDS and COVID-19-ARDS patient whole blood smears match characteristics of NET-formation with mitochondrial DNA (mtDNA) and intact cell membranes 24, 53 , and scanning electron microscopy images of NET-forming neutrophils 54 . Direct visualization of NET-forming neutrophils in patient blood smears documents intravascular events at time of blood draw, delineates NET-forming neutrophils as source of extruded DNA, and gives insight into pathophysiological context of different NET-subtypes. The algorithm-based automated quantitation of NET-forming neutrophils provides an objective quantitative method for direct morphological identification of NET-forming neutrophils in fixed patient blood smears, thus overcoming the limitation of nonquantitative visualization of IF-stained NETs 55 . These observations are supported by markedly elevated ratio of plasma mitochondrial DNA (mtDNA) to nuclear DNA (nDNA) copy-number in ARDS samples. Since elevated Table 1 . Spearman rank correlation matrix analysis: ARDS. n = 19 subjects ARDS, all cause [pre-COVID19 pandemic]. CBC-differential NLR, neutrophil lymphocyte ratio calculated from ratio of absolute neutrophil to absolute lymphocyte counts. Flow cytometry parameters #DEspR+ CD11b+ Ns, total number (#) in K/μL of DEspR+ C11b + neutrophils (Ns); %DEspR + CD11b + Ns, % of DEspR + CD11b + neutrophils among total (CD11b+/−) neutrophils; %DEspR + CD11b + [Ns + Ms], sum of the % of DEspR + CD11b + neutrophils and monocytes; %DEspR + CD11b + Ms, %DEspR + CD11b + monocytes among total (CD11b+/−) monocytes; %DEspR+CD11b+ Ls, %DEspR+CD11b+ lymphocytes among total (CD11b+/−) lymphocytes; #DEspR(−) Ns, total number (#) in K/µL of DEspR(−) neutrophils (Ns). cytoplast HI , cytoplasts with high SSC or granularity; cytoplast LO , low SSC or low granularity cytoplast; Plasma biomarkers Plasma levels of ET1, endothelin-1, IL-6, interleukin-6; MPO, myeloperoxidase levels; sC5b9, soluble complement terminal C5b9-complex; and mt/nucl DNA, ratio of mitochondrial DNA copy number to nuclear DNA copy number. Clinical measures of ARDS severity ICU-free days by day 28 = [28 minus # ICU days] with NonSurvivors = [− 1] and Survivors > 28 ICU-days = 0; S/F ratio, SpO2/FiO2 ratio as a measure of hypoxemia severity; SOFA, Sequential Organ Failure Assessment score; t1-SOFA, SOFA score on day of flow cytometry analysis; t2-SOFA, SOFA score at end of ICU stay. Statistical analysis Spearman Rank Order Correlation coefficient rho (r) effect size: strong r 0.6-0.79; very strong r 0.8-1.0. Data points are peak values for subjects with multiple FCM analyses. Spearman Rank Correlation Coefficient (rho, r > 0.61, p < 0.05, has Power > 0.8 with n = 19; significant Spearman rho with power 0.8 highlighted in italics; significant Spearman rho but power < 0.8 in bold. www.nature.com/scientificreports/ mtDNA released by necrotic cells is observed in ARDS 56 and COVID-19-ARDS 57 , using mtDNA/nDNA ratio could help further dissect mtDNA-NET levels from cell necrosis which would exhibit a mtDNA/nDNA ratio < 1 given that mtDNA is less than 1% of nuclear DNA. Detection of the classic DEspR+MPO+ DNA-cloud, DEspR+CD11b+ NET-forming neutrophils with intact cell membranes, and long DNA-remnants with DEspR+CD11b+ microvesicles on the DNA strands in the same patient blood smear altogether indicate ongoing multiple intravascular NET-formation events in ARDS and COVID-19-ARDS. Multiple NET-subtypes are placed into pathophysiological context and are not due to confounders from different methods of analysis, sample source or procurement 53,58-60 . Relative pathophysiological relevance is supported by the significant correlation of intravascular DEspR+/CD11b+ NET-forming neutrophilcounts with multiple clinical measures of severity in COVID-19-ARDS, in contrast to non-correlation of NLR, IL-6, and sC5b9 61, 62 in this prospective observational study.

Additionally, the presence of DEspR+CD11b+ ~ 200 micron-long intravascular DNA-strands, multi-NETforming neutrophil clusters, and NET-DNA strands straddling RBCs, are concordant with prior reports in COVID-19-ARDS and ARDS 1, 10, 11, 60 . This combination of multiple intravascular NET-structures likely cause intravascular flow impedance leading to low-flow ischemia with or without micro-thromboses. Intravascular impedance to flow without microthrombi can account for persistence of low-flow or micro-ischemic events in different organs in severe ARDS and COVID-19-ARDS despite pharmacological thromboprophylaxis or antithrombotic treatment 63 .

Data showing that DEspR-inhibition leads to neutrophil apoptosis in ARDS patient and Rhesus macaque samples support DEspR as an actionable therapeutic target. Induction of apoptosis in dysregulated, apoptosisresistant neutrophil-subset(s) 64 implicated in progressive secondary tissue injury leading to ARDS-MOF 5 has been deduced to be a critical step towards initiation of resolution of excessive inflammation in ARDS 1 . Hence, targeted-inhibition of DEspR+ neutrophils with endpoint induction of neutrophil apoptosis presents a potential therapeutic approach with advantages. First, hu6g8-mediated induction of DEspR+ neutrophil apoptosis attains function-shutdown of neutrophil-complement reciprocal co-activation 65 , hence potentially breaking this self-sustaining pro-cell injury mechanism. Second, induction of DEspR+ neutrophil apoptosis without cell lysis provides a key step for efferocytosis 66 of the DEspR+ neutrophil subset. Thirdly, induction of apoptosis in DEspR+ neutrophils preempts progression to NET-formation, hence a potential therapeutic approach to preventing DEspR+ dysregulated NET-formation. This gains relevance when neutrophils are PADI4-negative, as Table 2 . Spearman Rank Correlation Coefficients: COVID-19-ARDS subjects requiring ventilator support. n = 11 subjects with COVID-19-ARDS needing ventilatory support. CBC-differential NLR, neutrophil lymphocyte ratio. Flow cytometry parameters #DEspR+ CD11b+ Ns, total number (#) in K/μL of DEspR+C11b+ neutrophils (Ns); %DEspR+CD11b+Ns, % of DEspR+CD11b+ neutrophils among total (CD11b+/−) neutrophils; %DEspR+ CD11b+ [Ns+Ms], sum of the % of DEspR+ CD11b+ neutrophils and monocytes; %DEspR+CD11b+ Ms, %DEspR+CD11b+ monocytes among total (CD11b+/−) monocytes; #DEspR(-) Ns, total number (#) in K/µL of DEspR(−) neutrophils (Ns). Immunofluorescence Cytology parameters #DEspR+CD11b+ NET-forming Ns: number of DEspR+CD11b+ NET-forming Ns (% DEspR+ CD11b+NET-forming Ns x total number of DEspR+CD11b+ Ns); Circularity index, circularity index as a quantitative measure of NET-forming neutrophils with extruded DNA resulting in irregular perimeters with decreased similarity to a circle: outline of DAPI+ DNA fluorescence closest to 'perfect circle' = 1. Threshold 0.8: a NET-forming neutrophil has < 0.8 circularity index. Clinical measures of ARDS severity ICU-free days by day 28 = [28 minus # ICU days] with nonSurvivors = [− 1] and Survivors > 28 ICU-days = 0; S/F ratio, SpO2 converted to PaO2/FiO2 ratio as a measure of hypoxemia severity; SOFA, Sequential Organ Failure Assessment score; t1-SOFA, SOFA score on day of flow cytometry analysis; t2-SOFA, SOFA score at end of ICU stay (or day prior to death or discharge). Statistical analysis Spearman Rank Order Correlation coefficient rho (r) effect size: strong r 0.6-0.79; very strong r 0.8-1.0. Data points are peak values for subjects with multiple FCM analyses. Spearman Correlation Coefficient r > 0.76, alpha < 0.05, Power > 0.8, n = 11 (italics); Spearman r > 0.6, alpha < 0.05, power 0.7 to 0.8 (bold). www.nature.com/scientificreports/ seen in 98.6% of neutrophils in the COVID-19-ARDS RNA-seq files, hence non-responders to PADI4-inhibitors of NET-formation. While more studies are needed to elucidate mechanisms, induction of DEspR+ neutrophil apoptosis by DEspR inhibition complies with prior delineation that stopping neutrophil-mediated tissue injury requires induction of neutrophil apoptosis 1,64 . Importantly, consideration for potential on-target side effects, especially in the context of acute kidney injury as part of multi-organ failure in ARDS, highlights known DEspR+ expression in human medullary tubular epithelial cells 67 . In the presence of immunoglobinuria, anti-DEspR antibody passing through the glomerulus could present potential on-target tubular epithelial effects, but unlikely as antibody functionality will be altered in the increasingly acidic and hyperosmotic milieu in the kidney medullary lumen. Equally important, the critical limitation of pleiotropic neutrophil inhibitors in the context of ongoing infections and risk for secondary infections as in ARDS and COVID-19-ARDS, the target-specific inhibition of DEspR+ rogue neutrophils will spare DEspR[-]CD11b+ activated neutrophil subsets, hence preserve neutrophil defense functions against infections pertinent to critically ill patients with ARDS or COVID-19-ARDS.

Altogether, data identify the DEspR+CD11b+ neutrophil-subset as a therapeutic target with the potential to break the feed-forward progression of neutrophil-mediated tissue injury in ARDS and COVID-19-ARDS, while preserving DEspR[-] neutrophil functions. Data provide foundational basis for further study.

Limitations. We acknowledge the limitations of prospective pilot observational studies with n = 19 ARDS and n = 11 COVID-19-ARDS, and n = 19 COVID-19 scRNA profiles. With a focus on the study of neutrophils, we did not evaluate other cells with cytotoxic capabilities. We acknowledge the inherent limitations of the study of critically ill patients with limited patient samples for study, and limitations in our COVID-19-ARDS whole blood samples treated with 4% PFA to inactivate SARS CoV2 virus [final 2% PFA], leading to non-availability of plasma samples to perform ELISA studies on biomarkers and MPO-DNA complexes of NET-remnants.

Study design. Different tasks in this interdisciplinary pilot observational study among different collaborators were compartmentalized in order to attain blinding of researchers during task-performance. The following tasks were compartmentalized: Study subjects. All subjects were identified in the ICU under study protocols approved by the Institutional Review Board (IRB) of Boston University (IRB H-36744). Each subject's legal authorized representative gave written informed consent for study participation in compliance with the Declaration of Helsinki.

We enrolled 19 ARDS patients in the pre-COVID-19 pandemic period, and 11 COVID-19 ARDS patients admitted to the intensive care unit (ICU) at Boston Medical Center. ARDS diagnosis was based on clinical diagnosis using the Berlin Definition. COVID-19 ARDS patients were ascertained as COVID-19 positive by SARS-CoV-2 PCR testing. Additional data were obtained prospectively from 16 COVID-19 ARDS patients to examine the time-course during ICU-hospitalization and correlation of other known markers with survival: For study of ARDS and COVID-19-ARDS patient samples, whole blood (3 or 6 mls) was collected via preexisting indwelling peripheral vascular lines into K2-EDTA vacutainer tubes (FisherScientific, MA) from patients hospitalized in the ICU at Boston Medical Center by the ICU-nurse. COVID-19 patient EDTA-anticoagulated blood samples were immediately fixed with one volume of 4% PFA. Both Non-COVID and COVID-19 blood samples were processed for flow cytometry analysis within 1 h from blood collection. Platelet poor plasma was isolated and frozen at − 80 °C for future testing within 2 h from blood draw. Blood smears were prepared within 1 h from blood draw.

At BUSM, EDTA-anticoagulated blood samples from non-COVID ARDS subjects (100 μl per tube, × 2-3 replicates) were processed for flow cytometry within 1-h from blood sampling. [See Supplementary Methods for detail] Flow cytometry buffer comprised of Hank's balanced salt solution plus 2% heat-inactivated FBS as blocking agent; staining antibodies: 10 μg/ml of AF-647 labeled hu6g8 mAb, or the corresponding human IgG4-AF647 isotype IgG4, and 2.5 μg/ml anti-CD11b-AF488 or the corresponding mouse IgG1 kappa isotype control, AF-488; staining done at 4 °C × 30 min with rotation and protected from light; after staining, cells were fixed in 1% PBS-buffered PFA pH 7.4 at 4 °C, followed by RBC lysis at RT. After final wash, stained cells were resuspended in 400 μl HBSS 2% FBS, filtered and analyzed on a BD LSR-II flow cytometer. Analysis was done using FloJo Flow Cytometry Analysis Software (www. FloJo. com). Controls used were: both fluorescence minus one (FMO) controls, both isotype controls, compensation beads for both staining antibodies to check labeled antibody quality.

For disinfected COVID-19 blood samples (2%PFA-fixed), samples were washed 3 times with 8 volumes of HBSS + 2% FBS to remove residual fixative prior to processing for flow cytometry as described above. Each test sample run in duplicates.

At BWH, EDTA-anticoagulated whole blood samples were processed 2-3 h from sampling and white blood cells were separated from RBCs via Inertial Microfluidic Separation validated previously for neutrophil characterization 44 Representative image at t-12 h from video-recorded live cell imaging of isolated white blood cells (WBCs) documented to have > 90% DEspR+ CD11b+neutrophils among all neutrophils , and exposed to 10 μg/ml hu6g8-AF568 labeled antibody at 4 °C × 20 min to eliminate non-specific cell uptake by macropinocytosis or endocytosis. DEspR+ (red circle) Rhesus-neutrophils, apoptotic cell budding (encircled ○), and Sytox Green (SytoxG)-positive membrane permeable ( ). (H) Representative t-12 h image of isotype hu-IgG4 AF568 shows minimal to no isotype-AF568 (red circle) uptake; constitutive apoptosis cell budding (encircled ○), SytoxGreen-positive staining in cells with loss of cell membrane integrity ( 

Methods for details.] At Fraunhofer ITEM, heparinized whole blood was stored on ice until processing and used within 1-h after collection. Whole blood (100 µl) samples were washed with 1 ml of ice cold assay buffer, and cells were incubated in 100 µl of assay buffer containing bacterial endotoxin lipopolysaccharide LPS (100 ng/ml; Escherichia coli serotype 0111:B4) or assay buffer as control for 1 h at 37 °C. The reaction was then stopped, cells washed, then resuspended and cells were stained with hu6g8-PE (10 µg/ml) and CD11b-FITC for 30 min on ice under constant stirring in the dark. Cells were washed to remove unbound antibodies, fixed for 10 min at 4 °C, followed by RBC lysis. The cell pellet was resuspended in 250 µl flow cytometry buffer and was analyzed within 2 h using a Beckman Coulter Navios 3L 10C flow cytometer and data analyzed using Beckman Coulter Kaluza 2.1 Software.

At BUSM, 100 μl EDTA-anticoagulated whole blood samples (n = 6) were exposed to 75-100 μg/ml LPS at 37 °C × 1-h, then subjected to FCM analysis as described above.

Plasma level analysis of biomarkers by ELISA and quantitation of mitochondrial DNA. Individual ELISA protocols were performed as per manufacturer's instructions with the following sample dilutions: For MPO ELISA (abcam cat# ab195212) plasma dilution 1:1000; for C5b9 ELISA (MyBioSource cat# MBS2021557) plasma dilution 1:100; for IL-6 ELISA (Abcam cat# ab46027) plasma dilution 1:2; for ET1 ELISA (abcam cat# ab133030) plasma dilution 1:2.

To compare the levels of mitochondrial to nuclear DNA in human plasma samples we used the Nova-QUANT™ Human Mitochondrial to Nuclear DNA Ratio Kit (SIGMA-Aldrich cat# 72,620-1KIT) as per manufacturer's instructions. The kit measures the mtDNA copy number to that of nuclear DNA by Real-Time PCR of specific mitochondrial and nuclear genes optimized for equivalent amplification. Plasma DNA was isolated from 200 μl of plasma using the Quick-cfDNA Serum & Plasma Kit (Zymo Research, cat# D4076) as per manufacturer's instruction.

Immunofluorescence staining of NET-forming neutrophils. Blood smears were prepared by capillary action from EDTA anticoagulated whole blood (10 μL) samples on a Superfrost Plus Microscope slide (Fisher Scientific, cat# 12-550-15) within 1-h from blood sampling. Blood smears were air dried for 10 min then fixed with 100% Methanol (chilled to − 20 °C) for 10 min. Fixed slides were stored dry in − 20 °C freezer for future immunostaining. Immunofluorescence (IF)-staining to detect NET-forming neutrophils was done as described 68 , with custom modifications. We used anti-DEspR hu6g8 and anti-CD11b, as well as anti-DEspR and anti-MPO antibodies-conjugated to fluorophores (AF568, or AF488) for direct pair-wise immunostaining; DAPI for DNA detection. Chilled methanol fixation and permeabilization allowed fixation within 1 h from blood draw, eliminating need for paraformaldehyde and saponin. PBS with 5% FBS was used as blocking and binding solution for primary antibodies.

Fixed cell imaging of blood smears for quantiation of NET-forming neutrophils. Immunofluorescence imaging was performed as contract research service at Nikon Imaging Laboratory (Cambridge MA). Slides were imaged with a Nikon Ti2-E Widefield microscope equipped with a Plan Apo λ 20 × objective and Spectra LED light source and controlled by NIS-Elements. Briefly, an automated, JOBS routine in NIS-Elements Ex-vivo anti-DEspR treatment of ARDS patient blood samples. One ml of freshly obtained blood samples were incubated overnight at 37 °C with or without anti-DEspR mAb (hu6g8 at 100 μg/ml). After incubation half of the samples were subjected to FACS analysis as described above and the other half was processed for plasma isolation. Plasma MPO and C5b-9 levels were determined with corresponding ELISA kits as described above. 

The counter-intuitive role of the neutrophil in the acute respiratory distress syndrome

Acute respiratory distress syndrome

COVID-19: In the eye of the cytokine storm

Neutrophils in the initiation and resolution of acute pulmonary inflammation: understanding biological function and therapeutic potential

Neutrophils in development of multiple organ failure in sepsis

Immunologic alterations and the pathogenesis of organ failure in the ICU

New insights into neutrophil extracellular traps: Mechanisms of formation and role in inflammation

Neutrophil-to-lymphocyte ratio as a prognostic marker in acute respiratory distress syndrome patients: A retrospective study

Prognostic implications of neutrophil-lymphocyte ratio in COVID-19

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

Neutrophil extracellular traps and by-products play a key role in COVID-19: Pathogenesis, risk factors, and therapy

COVID-19 severity correlates with airway epithelium-immune cell interactions identified by single-cell analysis

Elevated calprotectin and abnormal myeloid cell subsets discriminate severe from Mild COVID-19

Induction of alarmin S100A8/A9 mediates activation of aberrant neutrophils in the pathogenesis of COVID-19

Severe COVID-19 is marked by a dysregulated myeloid cell compartment

Understanding a heterogeneous syndrome

Acute respiratory distress syndrome neutrophils have a distinct phenotype and are resistant to phosphoinositide 3-kinase inhibition

Humanized anti-DEspR IgG4 S228P antibody increases overall survival in a pancreatic cancer stem cellxenograft peritoneal carcinomatosis rat nu/nu model

Mcl-1 expression in human neutrophils: regulation by cytokines and correlation with cell survival

Neutrophil apoptosis: A target for enhancing the resolution of inflammation

Embryonic lethality in Dear gene deficient mice: New player in angiogenesis

Endothelin-1 in adult respiratory distress syndrome

Endothelin-1 enhances neutrophil adhesion to human coronary artery endothelial cells: role of ET(A) receptors and platelet-activating factor

Viable neutrophils release mitochondrial DNA to form neutrophil extracellular traps

NETosis: How vital is it

Confirmation of translatability and functionality certifies the dual endothelin1/VEGFsp receptor (DEspR) protein

Red cell DAMPs and inflammation

S100A8/A9 in inflammation

Pattern recognition receptor-dependent mechanisms of acute lung injury

LPS activation of Toll-like receptor 4 signals CD11b/CD18 expression in neutrophils

Neutrophil-mediated regulation of innate and adaptive immunity: The role of myeloperoxidase

Myeloperoxidase: molecular mechanisms of action and their relevance to human health and disease

Understanding the roles of cytokines and neutrophil activity and neutrophil apoptosis in the protective versus deleterious inflammatory response in pneumonia

How COVID-19 induces cytokine storm with high mortality

Acute lung injury: A clinical and molecular review

Impaired efferocytosis and neutrophil extracellular trap clearance by macrophages in ARDS

Neutrophil apoptosis in the acute respiratory distress syndrome

Identification of key signaling pathways induced by SARS-CoV2 that underlie thrombosis and vascular injury in COVID-19 patients

ICU-free days as a more sensitive primary outcome for clinical trials in critically ill pediatric patients

Patrolling monocytes promote intravascular neutrophil activation and glomerular injury in the acutely inflamed glomerulus

Neutrophil-to-lymphocyte ratio as an independent risk factor for mortality in hospitalized patients with COVID-19

The emerging role of neutrophil extracellular traps in severe acute respiratory syndrome coronavirus 2 (COVID-19)

SARS-CoV-2-triggered neutrophil extracellular traps mediate COVID-19 pathology

Neutrophil cytoplasts induce T H 17 differentiation and skew inflammation toward neutrophilia in severe asthma

Leukocyte function assessed via serial microlitre sampling of peripheral blood from sepsis patients correlates with disease severity

Of mice and not men: Differences between mouse and human immunology

DEspR roles in tumor vasculo-angiogenesis, invasiveness, CSC-survival and anoikis resistance: A 'common receptor coordinator' paradigm

Neutrophil heterogeneity as therapeutic opportunity in immune-mediated disease

High levels of S100A8/A9 proteins aggravate ventilator-induced lung injury via TLR4 signaling

Elevated serum levels of S100A8/A9 and HMGB1 at hospital admission are correlated with inferior clinical outcomes in COVID-19 patients

ADAR1 is involved in the development of microvascular lung injury

Endothelin-1 in acute lung injury and the adult respiratory distress syndrome

Untangling "NETosis" from NETs

Neutrophil extracellular traps in fatal COVID-19-associated lung injury. Dis. Mark. 2021, 5566826

Presence of neutrophil extracellular traps and citrullinated histone H3 in the bloodstream of critically ill patients

Prognostic value of plasma mitochondrial DNA in acute respiratory distress syndrome (ARDS): a single-center observational study

Circulating mitochondrial DNA is an early indicator of severe illness and mortality from COVID-19

An emerging role for neutrophil extracellular traps in noninfectious disease

Detection, visualization, and quantification of neutrophil extracellular traps (NETs) and NET Markers

To NET or not to NET: Current opinions and state of the science regarding the formation of neutrophil extracellular traps

Systemic complement activation is associated with respiratory failure in COVID-19 hospitalized patients

Complement activation in patients with COVID-19: A novel therapeutic target

Confirmation of the high cumulative incidence of thrombotic complications in critically ill ICU patients with COVID-19: An updated analysis

Neutrophil apoptosis: Relevance to the innate immune response and inflammatory disease

Complement alternative pathway acts as a positive feedback amplification of neutrophil activation

Modulation of neutrophil apoptosis and the resolution of inflammation through β2 integrins

Association of ATP1A1 and dear single-nucleotide polymorphism haplotypes with essential hypertension: sexspecific and haplotype-specific effects

Identification of neutrophil extracellular traps in the blood of patients with systemic inflammatory response syndrome

All funding agencies did not participate in the design, conception, data acquisition, analysis or interpretation of data, or manuscript preparation.

Boston University holds awarded and pending patents on DEspR. VLMH and NRO are co-inventors filed by Boston University. These patents comprise the option granted for exclusive license to NControl Therapeutics, Inc. VLMH, NRO are scientific co-founders of NControl Therapeutics, and paid consultants with equity in NControl Therapeutics, Inc. NControl Therapeutics was not involved in the design, conception, data interpretation, or manuscript preparation. All other co-authors have no competing interests. JNG: Consultant for NControl, Cayuga, CSL Behring, Alexion, Pfizer, Takeda. RMB: Member of Advisory Boards for Merck and Genentech. Reprints and permissions information is available at www.nature.com/reprints.

Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.