Lancet Microbe：新冠病毒感染相关肺曲霉菌病下呼吸道单细胞RNA测序与中性粒细胞细胞外陷阱

Abstract

Background: COVID-19-associated pulmonary aspergillosis (CAPA) is a severe superinfection with the fungus Aspergillus affecting patients who are critically ill with COVID-19. The pathophysiology and the role of neutrophil extracellular traps (NETs) in this infection are largely unknown. We aimed to characterise the immune profile, with a focus on neutrophils and NET concentrations, of critically ill patients with COVID-19, with or without CAPA.

背景：COVID-19相关的肺曲霉病（CAPA）是一种严重的超级感染，由曲霉菌引起，影响重症COVID-19患者。目前对这种感染的病理生理学和中性粒细胞外陷阱（NETs）的作用知之甚少。我们的目标是描述重症COVID-19患者（有或没有CAPA）的免疫特征，重点关注中性粒细胞和NETs浓度。

Methods: We conducted a single-centre, retrospective, observational study in two patient cohorts, both recruited at University Hospitals Leuven, Belgium. We included adults aged 18 years or older who were admitted to the intensive care unit because of COVID-19 between March 31, 2020, and May 18, 2021, and who were included in the previous Contagious trial (NCT04327570). We investigated the immune cellular landscape of CAPA versus COVID-19 only by performing single-cell RNA sequencing (scRNA-seq) on bronchoalveolar lavage fluid. Bronchoalveolar lavage immune cell fractions were compared between patients with CAPA and patients with COVID-19 only. Additionally, we determined lower respiratory tract NET concentrations using biochemical assays in patients aged 18 years and older who were admitted to the intensive care unit because of severe COVID-19 between March 15, 2020, and Dec 31, 2021, for whom bronchoalveolar lavage was available in the hospital biobank. Bronchoalveolar lavage NET concentrations were compared between patients with CAPA and patients with COVID-19 only and integrated with existing data on immune mediators in bronchoalveolar lavage and 90-day mortality.

方法：我们在比利时鲁汶大学医院进行了一项单中心、回顾性、观察性研究，包括两个患者队列。我们纳入了2020年3月31日至2021年5月18日期间因COVID-19入住重症监护室的18岁及以上成人，他们之前参加了Contagious试验（NCT04327570）。我们通过在支气管肺泡灌洗液上进行单细胞RNA测序（scRNA-seq）来研究CAPA与仅COVID-19的免疫细胞景观。比较了CAPA患者和仅COVID-19患者的支气管肺泡灌洗免疫细胞分数。此外，我们使用生化试验测定了2020年3月15日至2021年12月31日期间因严重COVID-19入住重症监护室的18岁及以上患者（医院生物库中有支气管肺泡灌洗液可用）的下呼吸道NET浓度。比较了CAPA患者和仅COVID-19患者的支气管肺泡灌洗NET浓度，并将其与支气管肺泡灌洗中的免疫介质和90天死亡率的现有数据整合。

Findings: We performed scRNA-seq of bronchoalveolar lavage on 43 samples from 39 patients, of whom 36 patients (30 male and six female; 14 with CAPA) were included in downstream analyses. We performed bronchoalveolar lavage NET analyses in 59 patients (46 male and 13 female), of whom 26 had CAPA. By scRNA-seq, patients with CAPA had significantly lower neutrophil fractions than patients with COVID-19 only (16% vs 33%; p=0·0020). The remaining neutrophils in patients with CAPA preferentially followed a hybrid maturation trajectory characterised by expression of genes linked to antigen presentation, with enhanced transcription of antifungal effector pathways. Patients with CAPA also showed depletion of mucosal-associated invariant T cells, reduced T helper 1 and T helper 17 differentiation, and transcriptional defects in specific aspects of antifungal immunity in macrophages and monocytes. We observed increased formation of NETs in patients with CAPA compared with patients with COVID-19 only (DNA complexed with citrullinated histone H3 median 15 898 ng/mL [IQR 4588-86 419] vs 7062 ng/mL [775-14 088]; p=0·042), thereby explaining decreased neutrophil fractions by scRNA-seq. Low bronchoalveolar lavage NET concentrations were associated with increased 90-day mortality in patients with CAPA.

发现：我们对39名患者的43个样本进行了scRNA-seq，其中36名患者（30名男性和6名女性；14名有CAPA）被包括在下游分析中。我们在59名患者（46名男性和13名女性）中进行了支气管肺泡灌洗NET分析，其中26名有CAPA。通过scRNA-seq，我们发现CAPA患者的中性粒细胞分数显著低于仅COVID-19患者（16%对33%；p=0.0020）。CAPA患者中剩余的中性粒细胞更倾向于遵循一种混合成熟轨迹，其特征是表达与抗原呈递相关的基因，并增强了抗真菌效应途径的转录。CAPA患者还表现出粘膜相关恒定T细胞的耗竭，减少了T辅助1和T辅助17的分化，以及巨噬细胞和单核细胞中特定抗真菌免疫方面的转录缺陷。我们观察到与仅COVID-19患者相比，CAPA患者中NETs的形成增加（与瓜氨酸化组蛋白H3复合的DNA中位数为15,898 ng/mL [IQR 4,588-86,419] 对 7,062 ng/mL [775-14,088]；p=0.042），从而解释了scRNA-seq中性粒细胞分数的降低。低支气管肺泡灌洗NET浓度与CAPA患者90天死亡率的增加有关。

Interpretation: Qualitative and quantitative disturbances in monocyte, macrophage, B-cell, and T-cell populations could predispose patients with severe COVID-19 to develop CAPA. Hybrid neutrophils form a specialised response to CAPA, and an adequate neutrophil response to CAPA is a major determinant for survival in these patients. Therefore, measuring bronchoalveolar lavage NETs could have diagnostic and prognostic value in patients with CAPA. Clinicians should be wary of aspergillosis when using immunomodulatory therapy that might inhibit NETosis to treat patients with severe COVID-19.

解释：单核细胞、巨噬细胞、B细胞和T细胞群体的定性和定量干扰可能使重症COVID-19患者易发生CAPA。混合中性粒细胞形成对CAPA的专门反应，适当的中性粒细胞反应是这些患者生存的主要决定因素。因此，测量支气管肺泡灌洗NETs可能对CAPA患者具有诊断和预后价值。在使用可能抑制NETosis以治疗重症COVID-19患者的免疫调节疗法时，临床医生应对曲霉病保持警惕。

Funding: Research Foundation Flanders, KU Leuven, UZ Leuven, VIB, the Fundação para a Ciência e a Tecnologia, the European Regional Development Fund, la Caixa Foundation, the Flemish Government, and Horizon 2020

资金来源：佛兰德研究基金会、KU Leuven、UZ Leuven、VIB、科学与技术基金会、欧洲区域发展基金、la Caixa基金会、佛兰德政府和地平线2020计划。