Herrera Victoria L M, Takahashi Courtney E, Nguyen Mai Q, Mosaddeghi Julie Z, Denis Ridiane, Greer David M, Ruiz-Opazo Nelson
Whitaker Cardiovascular Institute and Department of Medicine, Boston University School of Medicine, Boston, MA, United States.
Department of Neurology, Boston Medical Center and Boston University School of Medicine, Boston, MA, United States.
Front Neurol. 2022 Jul 25;13:935579. doi: 10.3389/fneur.2022.935579. eCollection 2022.
Cumulative 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.
In 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 () 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).
We detected strong correlations [(n = 13) > 0.71, power > 0.8, Bonferroni corrected < 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: [(n = 13) 0.94, = 10]. In contrast, plasma biomarkers tested were elevated except for MPO but exhibited no correlations in this pilot study.
Strong 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 analysis of patients with sICH neutrophils as a translational milestone to refine hypotheses between preclinical and clinical studies.
越来越多的临床、细胞和分子证据支持中性粒细胞在自发性脑出血（sICH）继发性脑损伤中所起的作用。然而，由于全身性抑制中性粒细胞是有害的，因此识别与sICH严重程度相关的可靶向“ rogue”中性粒细胞亚群是关键。
在一项针对自愿参与的sICH患者的前瞻性观察性初步研究中，我们对全血进行免疫分型，以评估循环中性粒细胞标志物（脑出血症状出现后约3天）：（a）通过流式细胞术检测DEspR±CD11b±中性粒细胞，（b）通过免疫荧光细胞学检测DEspR±CD11b±中性粒细胞胞外诱捕网（NET）形成中性粒细胞，以及（c）中性粒细胞与淋巴细胞比率（NLR）。使用经Bonferroni校正的Spearman等级相关性（），我们评估了中性粒细胞标志物与sICH严重程度的当日临床和神经影像学参数、脑出血指数评分、90天改良Rankin量表（mRS）评分以及潜在的相互关系。作为对照，我们评估了sICH中当日升高的血浆生物标志物：白细胞介素-6/IL-6、髓过氧化物酶/MPO、可溶性末端补体复合物/sC5b-9、内皮素-1/ET-1以及线粒体/核DNA比率（mt/nDNA比率）。
我们检测到所有三种中性粒细胞标志物与90天mRS评分之间存在强相关性[（n = 13）> 0.71，检验效能> 0.8，经Bonferroni校正的< 0.05]，DEspR + CD11b +中性粒细胞计数、NLR与血肿周围水肿（PHE）体积之间以及DEspR + CD11b + NET形成中性粒细胞计数与脑实质内出血（IPH）体积之间存在差异相关性。只有DEspR + CD11b +中性粒细胞计数与脑出血指数评分、当日格拉斯哥昏迷量表（GCS）评分、NLR以及NET形成中性粒细胞计数之间存在强相关性。脑出血指数评分与三种中性粒细胞标志物之和表现出最高相关性：[（n = 13）0.94，= 10]。相比之下，在本初步研究中，除MPO外，所检测的血浆生物标志物均升高，但未表现出相关性。
与多种sICH严重程度指标、NET形成和NLR的强相关性表明，DEspR + CD11b +中性粒细胞是sICH中一个假定的“ rogue”中性粒细胞亚群。三种中性粒细胞标志物之和与脑出血指数评分与90天mRS结局之间更强的相关性强化了早期中性粒细胞介导的继发性脑损伤是sICH患者结局的关键决定因素这一观点。总之，这些数据为正式研究DEspR + CD11b +中性粒细胞亚群作为sICH中中性粒细胞驱动的继发性脑损伤的潜在可操作生物标志物提供了基础。数据还表明，对sICH中性粒细胞患者的分析是完善临床前和临床研究之间假设的一个转化里程碑。
