DataSheet_1_Multiplex protein profiling of bronchial aspirates reveals disease-, mortality- and respiratory sequelae-associated signatures in critically ill patients with ARDS secondary to SARS-CoV-2 infection.docx

IntroductionBronchial aspirates (BAS) obtained during invasive mechanical ventilation (IMV) constitutes a useful tool for molecular phenotyping and decision making.
AimTo identify the proteomic determinants associated with disease pathogenesis, all-cause mortality and respiratory sequelae in BAS samples from critically ill patients with SARS-CoV-2-induced ARDS
MethodsMulticenter study including 74 critically ill patients with COVID-19 and non-COVID-19 ARDS. BAS were obtained by bronchoaspiration after IMV initiation. Three hundred sixty-four proteins were quantified using proximity extension assay (PEA) technology. Random forest models were used to assess predictor importance.
ResultsAfter adjusting for confounding factors, CST5, NADK, SRPK2 and TGF-α were differentially detected in COVID-19 and non-COVID-19 patients. In random forest models for COVID-19, CST5, DPP7, NADK, KYAT1 and TYMP showed the highest variable importance. In COVID-19 patients, reduced levels of ENTPD2 and PTN were observed in nonsurvivors of ICU stay, even after adjustment. AGR2, NQO2, IL-1α, OSM and TRAIL showed the strongest associations with in-ICU mortality and were used to construct a protein-based prediction model. Kaplan-Meier curves revealed a clear separation in mortality risk between subgroups of PTN, ENTPD2 and the prediction model. Cox regression models supported these findings. In survivors, the levels of FCRL1, NTF4 and THOP1 in BAS samples obtained during the ICU stay correlated with lung function (i.e., DLCO levels) 3 months after hospital discharge. Similarly, Flt3L and THOP1 levels were correlated with radiological features (i.e., TSS). These proteins are expressed in immune and nonimmune lung cells. Poor host response to viral infectivity and an inappropriate reparative mechanism seem to be linked with the pathogenesis of the disease and fatal outcomes, respectively.
ConclusionBAS proteomics identified novel factors associated with the pathology of SARS-CoV-2-induced ARDS and its adverse outcomes. BAS-based protein testing emerges as a novel tool for risk assessment in the ICU.

引言
有创机械通气（invasive mechanical ventilation, IMV）期间采集的支气管抽吸物（bronchial aspirates, BAS）是开展分子表型分析与临床决策的实用工具。

研究目的
本研究旨在明确重症新型冠状病毒（severe acute respiratory syndrome coronavirus 2, SARS-CoV-2）诱导的急性呼吸窘迫综合征（acute respiratory distress syndrome, ARDS）患者支气管抽吸物样本中，与疾病发病机制、全因死亡率及呼吸后遗症相关的蛋白质组学决定因素。

研究方法
本研究为多中心研究，纳入74例重症新冠病毒感染与非新冠病毒感染相关ARDS患者。于有创机械通气启动后，通过支气管抽吸术采集BAS样本。采用邻位延伸分析（proximity extension assay, PEA）技术对364种蛋白质进行定量检测。使用随机森林模型评估预测因子的重要性。

研究结果
校正混杂因素后，在新冠病毒感染与非新冠病毒感染患者的样本中，CST5、NADK、SRPK2及转化生长因子-α（transforming growth factor-α, TGF-α）的表达水平存在显著差异。针对新冠病毒感染患者的随机森林模型显示，CST5、DPP7、NADK、KYAT1及TYMP的变量重要性得分最高。在新冠病毒感染患者中，即使校正混杂因素后，ICU住院非存活者的ENTPD2与PTN水平均显著降低。AGR2、NQO2、白细胞介素-1α（interleukin-1α, IL-1α）、抑瘤素M（oncostatin M, OSM）及肿瘤坏死因子相关凋亡诱导配体（tumor necrosis factor-related apoptosis-inducing ligand, TRAIL）与ICU住院死亡率的相关性最强，基于此构建了蛋白质组学预测模型。卡普兰-迈耶（Kaplan-Meier）曲线显示，基于PTN、ENTPD2及该预测模型划分的亚组间死亡率风险存在显著区分度。Cox比例风险回归模型验证了上述结果。在存活患者中，ICU住院期间采集的BAS样本中FCRL1、NTF4及THOP1的水平，与出院3个月后的肺功能指标（即肺一氧化碳弥散量DLCO）呈显著相关。类似地，Flt3L与THOP1的水平与影像学特征（即TSS）相关。上述蛋白质在免疫与非免疫肺细胞中均有表达。宿主对病毒感染的应答不足与不适切的修复机制，分别与疾病发病机制及不良预后密切相关。

结论
支气管抽吸物蛋白质组学分析筛选出了与SARS-CoV-2诱导的ARDS病理进程及不良预后相关的新型分子标志物。基于BAS的蛋白质检测有望成为ICU内风险评估的新型工具。