Supplementary Material for: Impaired Degradation of Neutrophil Extracellular Traps: A Possible Severity Factor of Elderly Male COVID-19 Patients

Neutrophil extracellular traps (NETs) have been described as a potential trigger of severe COVID-19. NETs are known as extracellular DNA fibers released by neutrophils in response to infection. If the host is unable to balance efficient clearance of NETs by dornases (DNases), detrimental consequences occur. Elevated levels of NETs in COVID-19 patients are associated with higher risk of morbid thrombotic complications. Here, we studied the level of NET markers and DNase activity in a cohort of COVID-19 patients compared to healthy controls. Our data confirmed an increased level of NET markers in the plasma of COVID-19 patients, with a higher level in male compared to female patients. At the same time, there was an increased DNase activity detectable in COVID-19 patients compared to healthy controls. Importantly, there was a negative correlation of DNase activity with the age of male patients. The antimicrobial peptide LL-37, which is known to stabilize NETs against DNase degradation, is embedded in NETs upon severe acute respiratory syndrome coronavirus-2-infection. The LL-37 plasma level correlates with the NET-marker level in male COVID-19 patients, indicating a potential role of LL-37 in the risk of NET-associated thrombosis in male COVID-19 patients by stabilizing NETs against DNase degradation. In conclusion, our data identify two potential risk factors of elderly male patients which may lead to inefficient NET degradation and a subsequently higher risk of NET-associated thrombosis during COVID-19: reduced DNase activity and an increased LL-37 level.

中性粒细胞胞外陷阱（Neutrophil extracellular traps, NETs）已被证实为重症新型冠状病毒肺炎（COVID-19）的潜在触发因素。已知NETs是中性粒细胞应对感染时释放的胞外DNA纤维。若宿主无法通过脱氧核糖核酸酶（DNases）有效平衡NETs的清除，则会引发有害后果。COVID-19患者体内NETs水平升高，与病理性血栓并发症的更高发病风险相关。

本研究针对COVID-19患者队列，检测了NET标志物水平及DNase活性，并与健康对照组进行对比。研究数据证实，COVID-19患者血浆中的NET标志物水平升高，且男性患者的该指标水平高于女性患者。与此同时，相较于健康对照组，COVID-19患者体内可检测到的DNase活性亦有所升高。值得注意的是，男性患者的DNase活性与年龄呈负相关。

已知抗菌肽LL-37可稳定NETs以抵御DNase降解，在严重急性呼吸综合征冠状病毒2型（severe acute respiratory syndrome coronavirus-2）感染时，该肽会嵌入NETs结构中。在男性COVID-19患者中，血浆LL-37水平与NET标志物水平呈正相关，这提示LL-37可能通过稳定NETs抵御DNase降解，在男性COVID-19患者的NET相关血栓风险中发挥潜在作用。

综上，本研究数据明确了老年男性患者的两项潜在风险因素：二者可导致COVID-19期间NET降解效率低下，并进而升高NET相关血栓形成风险——一是DNase活性降低，二是LL-37水平升高。