Van Avondt Kristof, Schimmel Marein, Bulder Ingrid, van Mierlo Gerard, Nur Erfan, van Bruggen Robin, Biemond Bart J, Luken Brenda M, Zeerleder Sacha
Department of Immunopathology, Sanquin Research and Landsteiner Laboratory, Academic Medical Center Amsterdam, University of Amsterdam, Amsterdam, The Netherlands.
Institute of Experimental Pathology, Center for Molecular Biology of Inflammation, University Medical Center Münster, University of Münster, Münster, Germany.
Transfus Med Hemother. 2023 Mar 15;50(4):321-329. doi: 10.1159/000526760. eCollection 2023 Aug.
Neutrophils promote chronic inflammation and release neutrophil extracellular traps (NETs) that can drive inflammatory responses. Inflammation influences progression of sickle cell disease (SCD), and a role for NETs has been suggested in the onset of vaso-occlusive crisis (VOC). We aimed to identify factors in the circulation of these patients that provoke NET release, with a focus on triggers associated with hemolysis.
Paired serum and plasma samples during VOC and steady state of 18 SCD patients (HbSS/HbSβ-thal and HbSC/HbSβ-thal) were collected. Cell-free heme, hemopexin, and labile plasma iron have been measured in the plasma samples of the SCD patients. NETs formation by human neutrophils from healthy donors induced by serum of SCD patients was studied using confocal microscopy and staining for extracellular DNA using Sytox, followed by quantification of surface coverage using ImageJ.
Eighteen patients paired samples obtained during VOC and steady state were available (11 HbSS/HbSβ-thal and 7 HbSC/HbSβ-thal). We observed high levels of systemic heme and iron, concomitant with low levels of the heme-scavenger hemopexin in sera of patients with SCD, both during VOC and in steady state. In our in vitro experiments, neutrophils released NETs when exposed to sera from SCD patients. The release of NETs was associated with high levels of circulating iron in these sera. Although hemin triggered NET formation in vitro, addition of hemopexin to scavenge heme did not suppress NET release in SCD sera. By contrast, the iron scavengers deferoxamine and apotransferrin attenuated NET formation in a significant proportion of SCD sera.
Our results suggest that redox-active iron in the circulation of non-transfusion-dependent SCD patients activates neutrophils to release NETs, and hence, exerts a direct pro-inflammatory effect. Thus, we propose that chelation of iron requires further investigation as a therapeutic strategy in SCD.
中性粒细胞促进慢性炎症并释放中性粒细胞胞外诱捕网（NETs），后者可驱动炎症反应。炎症影响镰状细胞病（SCD）的进展，并且有人提出NETs在血管闭塞性危机（VOC）的发作中起作用。我们旨在确定这些患者循环中引发NET释放的因素，重点关注与溶血相关的触发因素。
收集了18例SCD患者（HbSS/HbSβ-地中海贫血和HbSC/HbSβ-地中海贫血）在VOC期间和稳定状态下的配对血清和血浆样本。对SCD患者的血浆样本中的游离血红素、血红素结合蛋白和不稳定血浆铁进行了测量。使用共聚焦显微镜和Sytox对细胞外DNA进行染色，研究了SCD患者血清诱导的健康供体人中性粒细胞形成NETs的情况，随后使用ImageJ对表面覆盖度进行定量。
获得了18例患者在VOC期间和稳定状态下的配对样本（11例HbSS/HbSβ-地中海贫血和7例HbSC/HbSβ-地中海贫血）。我们观察到，在VOC期间和稳定状态下，SCD患者血清中的全身血红素和铁水平较高，同时血红素清除剂血红素结合蛋白水平较低。在我们的体外实验中，中性粒细胞暴露于SCD患者的血清时会释放NETs。NETs的释放与这些血清中高水平的循环铁有关。尽管血红素在体外触发了NET的形成，但添加血红素结合蛋白清除血红素并不能抑制SCD血清中NET的释放。相比之下，铁螯合剂去铁胺和脱铁转铁蛋白在很大比例的SCD血清中减弱了NET的形成。
我们的结果表明，非输血依赖型SCD患者循环中的氧化还原活性铁激活中性粒细胞释放NETs，因此具有直接的促炎作用。因此，我们建议将铁螯合作为SCD的一种治疗策略需要进一步研究。
