Metzemaekers Mieke, Cambier Seppe, Blanter Marfa, Vandooren Jennifer, de Carvalho Ana Carolina, Malengier-Devlies Bert, Vanderbeke Lore, Jacobs Cato, Coenen Sofie, Martens Erik, Pörtner Noëmie, Vanbrabant Lotte, Van Mol Pierre, Van Herck Yannick, Van Aerde Nathalie, Hermans Greet, Gunst Jan, Borin Alexandre, Toledo N Pereira Bruna, Dos Sp Gomes Arilson Bernardo, Primon Muraro Stéfanie, Fabiano de Souza Gabriela, S Farias Alessandro, Proenca-Modena José Luiz, R Vinolo Marco Aurélio, Marques Pedro Elias, Wouters Carine, Wauters Els, Struyf Sofie, Matthys Patrick, Opdenakker Ghislain, Marques Rafael Elias, Wauters Joost, Gouwy Mieke, Proost Paul
Laboratory of Molecular Immunology Department of Microbiology, Immunology and Transplantation Rega Institute, KU Leuven Leuven Belgium.
Laboratory of Immunobiology Department of Microbiology, Immunology and Transplantation Rega Institute, KU Leuven Leuven Belgium.
Clin Transl Immunology. 2021 Apr 29;10(4):e1271. doi: 10.1002/cti2.1271. eCollection 2021.
Emerging evidence of dysregulation of the myeloid cell compartment urges investigations on neutrophil characteristics in coronavirus disease 2019 (COVID-19). We isolated neutrophils from the blood of COVID-19 patients receiving general ward care and from patients hospitalised at intensive care units (ICUs) to explore the kinetics of circulating neutrophils and factors important for neutrophil migration and activation.
Multicolour flow cytometry was exploited for the analysis of neutrophil differentiation and activation markers. Multiplex and ELISA technologies were used for the quantification of protease, protease inhibitor, chemokine and cytokine concentrations in plasma. Neutrophil polarisation responses were evaluated microscopically. Gelatinolytic and metalloproteinase activity in plasma was determined using a fluorogenic substrate. Co-culturing healthy donor neutrophils with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) allowed us to investigate viral replication in neutrophils.
Upon ICU admission, patients displayed high plasma concentrations of granulocyte-colony-stimulating factor (G-CSF) and the chemokine CXCL8, accompanied by emergency myelopoiesis as illustrated by high levels of circulating CD10, immature neutrophils with reduced CXCR2 and C5aR expression. Neutrophil elastase and non-metalloproteinase-derived gelatinolytic activity were increased in plasma from ICU patients. Significantly higher levels of circulating tissue inhibitor of metalloproteinase 1 (TIMP-1) in patients at ICU admission yielded decreased total MMP proteolytic activity in blood. COVID-19 neutrophils were hyper-responsive to CXCL8 and CXCL12 in shape change assays. Finally, SARS-CoV-2 failed to replicate inside human neutrophils.
Our study provides detailed insights into the kinetics of neutrophil phenotype and function in severe COVID-19 patients, and supports the concept of an increased neutrophil activation state in the circulation.
髓样细胞区室失调的新证据促使人们对2019冠状病毒病（COVID-19）中性粒细胞的特征进行研究。我们从接受普通病房护理的COVID-19患者以及入住重症监护病房（ICU）的患者血液中分离出中性粒细胞，以探索循环中性粒细胞的动力学以及对中性粒细胞迁移和激活重要的因素。
利用多色流式细胞术分析中性粒细胞分化和激活标志物。采用多重检测和酶联免疫吸附测定（ELISA）技术定量血浆中的蛋白酶、蛋白酶抑制剂、趋化因子和细胞因子浓度。通过显微镜评估中性粒细胞极化反应。使用荧光底物测定血浆中的明胶酶解和金属蛋白酶活性。将健康供体的中性粒细胞与严重急性呼吸综合征冠状病毒2（SARS-CoV-2）共同培养，以研究病毒在中性粒细胞中的复制情况。
入住ICU时，患者血浆中粒细胞集落刺激因子（G-CSF）和趋化因子CXCL8浓度较高，同时伴有应急性骨髓生成，表现为循环CD10水平升高，即CXCR2和C5aR表达降低的未成熟中性粒细胞增多。ICU患者血浆中的中性粒细胞弹性蛋白酶和非金属蛋白酶衍生的明胶酶解活性增加。入住ICU时患者循环中的金属蛋白酶组织抑制剂1（TIMP-1）水平显著升高，导致血液中总基质金属蛋白酶（MMP）的蛋白水解活性降低。在形状变化试验中，COVID-19中性粒细胞对CXCL8和CXCL12的反应性增强。最后，SARS-CoV-2未能在人类中性粒细胞内复制。
我们的研究详细深入地了解了重症COVID-19患者中性粒细胞表型和功能的动力学，并支持循环中中性粒细胞激活状态增加的概念。
