Supplementary Material for: Circulating Iron in Patients with Sickle Cell Disease Mediates the Release of Neutrophil Extracellular Traps

Introduction: 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. Methods: Paired serum and plasma samples during VOC and steady state of 18 SCD patients (HbSS/HbSβ0-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. Results: Eighteen patients paired samples obtained during VOC and steady state were available (11 HbSS/HbSβ0-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. Discussion: 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.

引言：中性粒细胞促进慢性炎症并释放中性粒细胞胞外陷阱（NETs），这些陷阱能够驱动炎症反应。炎症影响镰状细胞病（SCD）的进展，NETs在血管闭塞性危象（VOC）的发病机制中亦被提出具有作用。本研究旨在识别这些患者循环中的引发NET释放的因素，重点关注与溶血相关的触发因素。方法：收集了18名镰状细胞病（HbSS/HbSβ0-thal和HbSC/HbSβ+-thal）患者VOC期间和稳态时的血清和血浆样本对。在镰状细胞病患者的血浆样本中测量了无细胞血红素、血红素结合蛋白和可溶性血浆铁。通过共聚焦显微镜和Sytox染料对健康捐献者的人中性粒细胞诱导的NETs形成进行研究，并使用ImageJ进行表面覆盖率的量化。结果：在VOC期间和稳态状态下，可获得18对患者的样本（11例HbSS/HbSβ0-thal和7例HbSC/HbSβ+-thal）。我们观察到系统性血红素和铁的水平较高，同时镰状细胞病患者的血清中血红素清除剂血红素结合蛋白的水平较低。在我们的体外实验中，中性粒细胞在暴露于镰状细胞病患者的血清后释放NETs。NETs的释放与这些血清中循环铁的高水平相关。尽管体外实验中血红素触发了NETs的形成，但添加血红素结合蛋白以清除血红素并不能抑制镰状细胞病血清中的NETs释放。相反，铁清除剂去铁胺和铁蛋白清除剂在相当一部分镰状细胞病血清中减弱了NETs的形成。讨论：我们的结果表明，非输血依赖型镰状细胞病患者的循环中具有氧化还原活性的铁激活中性粒细胞释放NETs，从而产生直接的促炎作用。因此，我们建议铁螯合疗法作为镰状细胞病的治疗策略需要进一步研究。