“Rogue” [DEspR+CD11b+] neutrophil subset correlates with severity in spontaneous intracerebral hemorrhage

Abstract

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.

“Rogue” neutrophil-subset [DEspR+CD11b+/CD66b+] immunotype is an actionable therapeutic target for neutrophilic inflammation-mediated tissue injury – studies in human, macaque and rat LPS-inflammation models

“…Equally important, the observed subset-specific sparing of DEspR[-] neutrophils provides a mode-of-action for inherent safety: while DEspR+CD11b+ 'rogue' neutrophils are blocked, DEspR[-]CD11b+ neutrophils are 'spared' to carry on homeostatic neutrophil-mediated surveillance and defense mechanisms. These observations are concordant with prior observations that low DEspR+ neutrophil counts in ARDS (23) and ICH (34) patients are associated with recovery respectively, and that healthy donor neutrophils are DEspR[-] (23).…”

“…The consistent detection of the DEspR+ neutrophil-subset across three species in different LPS-induced neutrophilic inflammation models with increasing severity – 1) segmental neutrophilic inflammation and pulmonary-vascular barrier disruption model in human volunteers ( 33 ), 2) transient hypoxemia/ALI in macaques, and 3) high-mortality encephalopathy in rats – independently corroborate the neutrophil-subset with surface immunotype DEspR+CD11b+/CD66b+. These results confirm identity of this subset as detected in patients with ARDS, COVID-19-ARDS ( 23 ), and more recently, in spontaneous intracerebral hemorrhage (sICH) patients ( 34 ). Detection of DEspR+CD66b+ neutrophils, with differential cellular localization in healthy volunteer BALF 24h after segmental-LPS challenge is concordant with previous studies showing a] DEspR+CD11b+MPO+ neutrophils in intra-alveolar exudates with cell membrane, cytoplasmic and nuclear expression in postmortem lung tissue sections from ARDS patients ( 23 ), and DEspR+ tumor cells ( 31 ).…”

“…The demonstrations of efficacy of DEspR inhibition in vivo in two independent animal LPS-inflammatory models in reducing neutrophil-induced secondary tissue injury damage, 1) hypoxemia in macaques and 2) brain edema and encephalopathy in hypertensive rats, together indicate a pathophysiological role of the DEspR+ rogue neutrophil subset. More specifically, results support the hypothesis that LPS-induced DEspR+CD11b+ neutrophils contribute to the disruption of blood-tissue barriers, and provide experimental support for the strong correlation (Rs > 0.7) of DEspR+ “rogue” neutrophil-subset with SOFA-score in ARDS ( 23 ) and with 90-day modified Rankin Scale score for outcome in sICH patients ( 34 ).…”

“…The consistent detection of the DEspR+ neutrophil-subset across three species in different LPS-induced neutrophilic inflammation models with increasing severity -1) segmental day modified Rankin Scale score for outcome in sICH patients (34).…”

Circulating neutrophil extracellular trap (NET)-forming ‘rogue’ neutrophil subset, immunotype [DEspR+CD11b+], mediate multi-organ failure in COVID-19 - an observational study

“…'blood smears'. Cytology slides were prepared directly from whole blood within 1hr from blood draw in EDTA-anticoagulated samples (BMC) [19,23] and within 1-3hrs from blood draw from acid-citrate dextrose samples (MMC) in order to preserve, hence detect circulating neutrophils with fragile DNA-webs/strands, as first observed in activated NET-forming neutrophils ex vivo Statistical Analyses. Descriptive statistics and Spearman rank correlation analysis were performed using (PRISM 9.4, GraphPad).…”