Journal: Research Square

Article Title: Increased Neutrophil-Subset Associated With Severity/Mortality In ARDS And COVID19-ARDS Expresses The Dual Endothelin-1/VEGFsignal-Peptide Receptor (DEspR): An Actionable Therapeutic Target

doi: 10.21203/rs.3.rs-846250/v1

Figure Lengend Snippet: Correlation matrix analysis of DEspR+ neutrophils, clinical parameters and plasma biomarkers in ARDS and COVID19-ARDS. (A) Correlation matrix ARDS: Spearman Rank Correlation, n = 19 patients with ARDS diagnosis (Berlin definition) of cell surface DEspR+ expression levels (% or number #) in CD11b+ activated neutrophils (actNs), monocytes (actMs), and lymphocytes (actLs) and corresponding correlation with 1] clinical parameters of severity [ICU-free days at 28-days, defined as (28 - # of ICU-days) with ICU-death = −1, and > 28-days in the ICU = 0; PsO2/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 mitochondrial/nuclear DNA ratio (mt/nucl DNA), and 3] NETosis parameters: DEspR+ cytoplast levels with high or low granularity (SSC) on flow cytometry. See for specific values: rho, P-values with power > 0.8. (B) ARDS: neutrophil-to-lymphocyte ratio (NLR) between survivors (S) n = 12, (mean ± sd: 10.96 ± 5.4) and non-survivors (non-S) n = 6 (18.03 ± 11.21), Mann Whitney p = 0.33 (n.s). (C) ARDS: number (#) of cell-surface DEspR+CD11b+ neutrophils (Ns) detected in ARDS-survivors: n = 12, (mean ± sd: 0.8035 ± 0.8) vs non-survivors: n = 6 (8.1 ± 6.0); two-tailed Mann Whitney p = 0.0001 (***), effect size Hedges’ g with 4% correction: 2.03). (D) ARDS: Comparison of % of DEspR+CD11b+ neutrophils in ARDS: survivors: n = 13, (10.3 ± 10.0) vs non-survivors: n = 6, (48.2 ± 6.3); two-tailed Mann Whitney p < 0.0001 (****), effect size Hedges’ g with 4% correction: 4.03. (E) ARDS: Kaplan-Meir Survival curve analysis with threshold for DEspR+CD11b+ neutrophil-counts set at 3 K/μL whole blood as determined from . Log rank (Mantel-Cox) test Chi square 20.56, P < 0.0001, Hazard Ratio (Mantel-Haenszel) 90.5, 95% CI of ratio: 12.91 to 634.7. (F) Correlation matrix COVID19-ARDS: n = 11 (severe COVID19 requiring mechanical ventilation). Parameters as defined in , with addition of # of DEspR+CD11b+ NETosing neutrophils (D+actNETosing Ns), and circularity index of neutrophils (< 0.8 indicative of NETosing neutrophil, see ). (G) COVID19-ARDS: comparison of neutrophil-to-lymphocyte ratio (NLR) between survivors: n = 7 (mean ± sd: 10.7 ± 7.7) and non-survivors: n = 4 (55.3 ± 41.4). two-tailed Mann Whitney p = 0.0242 (*), effect size Hedge’s g with 4% correction: 1.73. (H) COVID19-ARDS: comparison of DEspR+CD11b+ neutrophil-counts (K/μL) in whole blood between survivors: n = 7 (mean ± sd: 3.8 ± 2.99) and non-Survivors: n = 4 (13.8 ± 7.9), two-tailed Mann Whitney p = 0.04 (*), effect size Hedge’s g with 4% correction: 1.82. (I) COVID19-ARDS: #DEspR+ NETosing neutrophil-counts (K/μL) whole blood, survivors: n = 7 (mean ± sd: 1.3 ± 1.29) and non-Survivors: n = 4 (10.0 ± 5.7). Two-tailed Mann Whitney p = 0.0121 (*), effect size Hedge’s g with 4% correction: 2.4.

Article Snippet: Correlations were calculated by using the Spearman Rank Order correlation test (GraphPad Prism v9.0.1) and power calculations determined by using SigmaPlot 11.0 software.

Techniques: Clinical Proteomics, Biomarker Discovery, Expressing, Flow Cytometry, MANN-WHITNEY, Two Tailed Test, Comparison

Journal: Research Square

Article Title: Increased Neutrophil-Subset Associated With Severity/Mortality In ARDS And COVID19-ARDS Expresses The Dual Endothelin-1/VEGFsignal-Peptide Receptor (DEspR): An Actionable Therapeutic Target

doi: 10.21203/rs.3.rs-846250/v1

Figure Lengend Snippet: Spearman Rank Correlation matrix analysis: COVID19-ARDS subjects requiring ventilator support.

Article Snippet: Correlations were calculated by using the Spearman Rank Order correlation test (GraphPad Prism v9.0.1) and power calculations determined by using SigmaPlot 11.0 software.

Techniques: Flow Cytometry, Immunofluorescence

Journal: Research Square

Article Title: Increased Neutrophil-Subset Associated With Severity/Mortality In ARDS And COVID19-ARDS Expresses The Dual Endothelin-1/VEGFsignal-Peptide Receptor (DEspR): An Actionable Therapeutic Target

doi: 10.21203/rs.3.rs-846250/v1

Figure Lengend Snippet: ( A ) Representative epifluorescence/DIC images of IF-stained blood smears from ARDS-non-survivor* ARDS-survivor, and non-ARDS critically ill patient in the ICU, DEspR+ NETosing neutrophils (dashed ○) and cytoplasts (➔). anuclear (DAPI−) red blood cells (RBCs) relief images in background. Bar = 20 μm. ( B ) Representative epifluorescence/DIC images of IF-stained blood smears from COVID19-ARDS non-survivor and survivor. IF-staining identical to panel A: inset: DEspR+ NETosing neutrophils prior to merged DAPI+DEspR+ image of NETosing neutrophils with extruded DNA-interconnections among neutrophils. Middle panel: triple-merged DEspR+CD11b+DAPI+ NETosing neutrophils with extruded DNA-interconnections. ( C ) Representative COVIDl9-ARDS-survivor showing minimal NETosing neutrophils. ( D-E ) Representative image of filamentous DEspR-bound extruded DNA networks in IF-stained blood smears from ARDS-patient ( D ) and COVID19-ARDS patient ( E ). ( F ) Analysis of circularity to distinguish NETosing neutrophil with low circularity from non-NETosing neutrophils with circularity index approaching 1.0. ( G ) Spearman rank correlation analysis of # DEspR+CD11b+ neutrophils and mean circularity index (average from n > 500 neutrophils per patient) in COVID19-ARDS patients (n = 11): negative correlation (r s = −0.782, P = 0.0064). ( H ) Flow cytometry analysis of FSC SSC and CD11b+ gated cytoplasts comparing levels in ARDS survivor (3.8%) vs ARDS non-survivor (29.77 %).

Article Snippet: Correlations were calculated by using the Spearman Rank Order correlation test (GraphPad Prism v9.0.1) and power calculations determined by using SigmaPlot 11.0 software.

Techniques: Staining, Flow Cytometry