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-IgG4S228P 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.

ObjectiveCumulative clinical, cellular, and molecular evidence reinforces the role of neutrophils in secondary brain injury in spontaneous intracerebral hemorrhage (sICH). However, since generalized neutrophil inhibition is detrimental, identification of targetable “rogue” neutrophil subsets associated with sICH severity is key.MethodsIn a pilot prospective observational study of consented patients with sICH, we immunotyped whole blood to assess circulating neutrophil markers (~day 3 after ICH symptoms onset): (a) DEspR±CD11b± neutrophils by flow cytometry, (b) DEspR±CD11b± neutrophil extracellular trap (NET)-forming neutrophils by immunofluorescence cytology, and (c) neutrophil-lymphocyte ratio (NLR). Using Spearman rank correlation (r) with Bonferroni correction, we assessed the association of neutrophil markers with same-day clinical and neuroimaging parameters of sICH severity, index ICH score, 90-day modified Rankin Scale (mRS) score, and potential interrelationships. As comparators, we assessed same-day plasma biomarkers elevated in sICH: interleukin-6/IL-6, myeloperoxidase/MPO, soluble-terminal complement complex/sC5b-9, endothelin-1/ET-1, and mitochondrial/nuclear DNA ratio (mt/nDNA ratio).ResultsWe detected strong correlations [r(n = 13) > 0.71, power > 0.8, Bonferroni corrected pB < 0.05] for all three neutrophil markers with 90-day mRS score, differentially for DEspR+CD11b+ neutrophil counts, and NLR with perihematomal edema (PHE) volume and for DEspR+CD11b+ NET-forming neutrophil counts with intraparenchymal hemorrhage (IPH)-volume. Only DEspR+CD11b+ neutrophil counts show a strong correlation with index ICH score, same-day Glasgow Coma Scale (GCS) score, and NLR and NET-forming neutrophil counts. The sum of the ICH score and three neutrophil markers exhibited the highest correlation: [r(n = 13) 0.94, pB = 10−5]. In contrast, plasma biomarkers tested were elevated except for MPO but exhibited no correlations in this pilot study.ConclusionStrong correlation with multiple sICH severity measures, NET formation, and NLR identifies DEspR+CD11b+ neutrophils as a putative “rogue” neutrophil subset in sICH. The even stronger correlation of the sum of three neutrophil markers and the index ICH score with 90-day mRS outcome reinforces early neutrophil-mediated secondary brain injury as a key determinant of outcome in patients with sICH. Altogether, data provide a basis for the formal study of the DEspR+CD11b+ neutrophil subset as a potential actionable biomarker for neutrophil-driven secondary brain injury in sICH. Data also show ex vivo analysis of patients with sICH neutrophils as a translational milestone to refine hypotheses between preclinical and clinical studies.

Background: Cumulative research show association of neutrophils and neutrophil extracellular traps (NETs) with poor outcomes in severe COVID-19. However, to date, no curative intent therapy has been identified to block neutrophil/NETs-mediated progression of multi-organ dysfunction. Because of emerging neutrophil heterogeneity, the study of subsets of circulating neutrophil-extracellular trap (NET)-forming neutrophils [NET+Ns] as mediators of multi-organ failure progression among patients with COVID-19 is critical to identification of therapeutic targets. Methods: We conducted a prospective observational study of circulating levels of CD11b+[NET+N] immunotyped for dual endothelin-1/signal peptide receptor, (DEspR±) expression by quantitative immunofluorescence-cytology and causal mediation analysis. In 36 consented adults hospitalized with mod-severe COVID-19, May to September 2020, we measured acute multi-organ failure via SOFA-scores and respiratory failure via SaO2/FiO2 (SF)ratio at time points t1 (average 5.5 days from ICU/hospital admission) and t2 (the day before ICU-discharge or death), and ICU-free days at day28 (ICUFD). Circulating absolute neutrophil counts (ANC) and [NET+N] subset-specific counts were measured at t1. Spearman correlation and causal mediation analyses were conducted. Results: Spearman correlation analyses showed correlations of t1-SOFA with t2-SOFA (rho rS=0.80) and ICUFD (rS=-0.76); circulating DEspR+[NET+Ns] with t1-SOFA (rS= 0.71), t2-SOFA (rS =0.62), and ICUFD (rS =-0.63), and ANC with t1-SOFA (rS=0.71), and t2-SOFA (rS=0.61). Causal mediation analysis identified DEspR+[NET+Ns] as mediator of 44.1% [95% CI:16.5,110.6] of the causal path between t1-SOFA (exposure) and t2-SOFA (outcome), with 46.9% [15.8,124.6] eliminated when DEspR+[NET+Ns] were theoretically reduced to zero. Concordantly, DEspR+[NET+Ns] mediated 47.1% [22.0,72.3%] of the t1-SOFA to ICUFD causal path, with 51.1% [22.8,80.4%] eliminated if DEspR+[NET+Ns] were reduced to zero. In patients with t1-SOFA >1, the indirect effect of a hypothetical treatment eliminating DEspR+[NET+Ns] projected a reduction of t2-SOFA by 0.98 [0.29,2.06] points and ICUFD by 3.0 [0.85,7.09] days. In contrast, there was no significant mediation of SF-ratio through DEspR+[NET+Ns], and no significant mediation of SOFA-score through ANC. Conclusions: Despite equivalent correlations, DEspR+[NET+Ns], but not ANC, mediated progression of multi-organ failure in acute COVID-19, and its hypothetical reduction is projected to improve ICUFD. These translational findings warrant further studies of DEspR+[NET+Ns] as potential patient-stratifier and actionable therapeutic target for multi-organ failure in COVID-19.

Background Cumulative research show association of neutrophils and neutrophil extracellular traps (NETs) with poor outcomes in severe COVID-19. However, to date, there is no curative intent therapy able to block neutrophil/NETs-mediated progression of multi-organ dysfunction. Because of emerging neutrophil heterogeneity, the study of subsets of circulating NET-forming neutrophils [NET + Ns] as mediators of multi-organ failure progression among patients with COVID-19 is critical to identification of therapeutic targets. Methods We conducted a prospective observational study of circulating levels of CD11b + [NET + N] immunotyped for dual endothelin-1/signal peptide receptor (DEspR ±) expression by quantitative immunofluorescence-cytology and causal mediation analysis. In 36 consented adults hospitalized with mod-severe COVID-19, May to September 2020, we measured acute multi-organ failure via SOFA-scores and respiratory failure via SaO2/FiO2 (SF)-ratio at time points t1 (average 5.5 days from ICU/hospital admission) and t2 (the day before ICU-discharge or death), and ICU-free days at day28 (ICUFD). Circulating absolute neutrophil counts (ANC) and [NET + N] subset-specific counts were measured at t1. Spearman correlation and causal mediation analyses were conducted. Results Spearman correlation analyses showed correlations of t1-SOFA with t2-SOFA (rho rS = 0.80) and ICUFD (rS = -0.76); circulating DEspR + [NET + Ns] with t1-SOFA (rS = 0.71), t2-SOFA (rS = 0.62), and ICUFD (rS = -0.63), and ANC with t1-SOFA (rS = 0.71), and t2-SOFA (rS = 0.61). Causal mediation analysis identified DEspR + [NET + Ns] as mediator of 44.1% [95% CI:16.5,110.6] of the causal path between t1-SOFA (exposure) and t2-SOFA (outcome), with 46.9% [15.8,124.6] eliminated when DEspR + [NET + Ns] were theoretically reduced to zero. Concordantly, DEspR + [NET + Ns] mediated 47.1% [22.0,72.3%] of the t1-SOFA to ICUFD causal path, with 51.1% [22.8,80.4%] eliminated if DEspR + [NET + Ns] were reduced to zero. In patients with t1-SOFA > 1, the indirect effect of a hypothetical treatment eliminating DEspR + [NET + Ns] projected a reduction of t2-SOFA by 0.98 [0.29,2.06] points and ICUFD by 3.0 [0.85,7.09] days. In contrast, there was no significant mediation of SF-ratio through DEspR + [NET + Ns], and no significant mediation of SOFA-score through ANC. Conclusions Despite equivalent correlations, DEspR + [NET + Ns], but not ANC, mediated progression of multi-organ failure in acute COVID-19, and its hypothetical reduction is projected to improve ICUFD. These translational findings warrant further studies of DEspR + [NET + Ns] as potential patient-stratifier and actionable therapeutic target for multi-organ failure in COVID-19.