Abstract：
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.
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.
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.
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.
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.
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.
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.
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.
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.
摘要：
背景：COVID-19相关的肺曲霉病（CAPA）是一种严重的曲霉真菌重叠感染，影响COVID-19危重患者。中性粒细胞胞外诱捕网（NETs）在这种感染中的病理生理学和作用尚不清楚。我们的目的是描述免疫特征，关注中性粒细胞和净浓度，患有或不患有CAPA的COVID-19危重患者。
方法：我们进行了单中心，回顾性,两个患者队列的观察性研究，都是在鲁汶大学医院招募的,比利时。我们纳入了年龄在18岁或以上的成年人，他们在2020年3月31日至2021年5月18日因COVID-19入住重症监护病房，并被纳入先前的传染性试验(NCT04327570)。我们仅通过对支气管肺泡灌洗液进行单细胞RNA测序（scRNA-seq），研究了CAPA与COVID-19的免疫细胞格局。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,我们在2020年3月15日至2021年12月31日因严重COVID-19而入住重症监护病房的18岁及以上患者中,通过生化试验测定了下呼吸道NET浓度,这些患者在医院生物库进行了支气管肺泡灌洗.比较了CAPA患者和仅COVID-19患者的支气管肺泡灌洗NET浓度，并结合了支气管肺泡灌洗中免疫介质的现有数据和90天死亡率。
结果：我们对39例患者的43个样本进行了支气管肺泡灌洗的scRNA-seq，其中36例患者(30例男性和6例女性;14例CAPA患者)被纳入下游分析.我们对59例患者（男性46例，女性13例）进行了支气管肺泡灌洗NET分析，其中26人患有CAPA。通过scRNA-seq，CAPA患者的中性粒细胞分数显著低于单纯COVID-19患者(16%vs33%;p=0·0020).CAPA患者的剩余中性粒细胞优先遵循杂交成熟轨迹，其特征是与抗原呈递相关的基因表达。抗真菌效应子途径的转录增强。CAPA患者还显示粘膜相关的不变T细胞的消耗，T辅助细胞1和T辅助细胞17分化减少，以及巨噬细胞和单核细胞抗真菌免疫特定方面的转录缺陷。我们观察到，与仅COVID-19患者相比，CAPA患者的NETs形成增加（与瓜氨酸化组蛋白H3复合的DNA中位数为15898ng/mL[IQR4588-86419]对7062ng/mL[775-14088]；p=0·042），从而解释了通过scRNA-seq减少的中性粒细胞分数。低支气管肺泡灌洗NET浓度与CAPA患者90天死亡率增加相关。
结论：单核细胞的定性和定量紊乱，巨噬细胞,B细胞,T细胞群可能使患有严重COVID-19的患者易患CAPA。混合中性粒细胞对CAPA形成专门的反应，和适当的中性粒细胞对CAPA的反应是这些患者生存的主要决定因素。因此,检测支气管肺泡灌洗NETs对CAPA患者具有诊断和预后价值。临床医生在使用可能抑制NETosis的免疫调节疗法治疗重症COVID-19患者时应警惕曲霉病。
背景：佛兰德斯研究基金会，KULeuven,UZLeuven,VIB,基金会，欧洲区域发展基金，laCaixa基金会，佛兰德政府,地平线2020
方法：我们进行了单中心，回顾性,两个患者队列的观察性研究，都是在鲁汶大学医院招募的,比利时。我们纳入了年龄在18岁或以上的成年人，他们在2020年3月31日至2021年5月18日因COVID-19入住重症监护病房，并被纳入先前的传染性试验(NCT04327570)。我们仅通过对支气管肺泡灌洗液进行单细胞RNA测序（scRNA-seq），研究了CAPA与COVID-19的免疫细胞格局。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,我们在2020年3月15日至2021年12月31日因严重COVID-19而入住重症监护病房的18岁及以上患者中,通过生化试验测定了下呼吸道NET浓度,这些患者在医院生物库进行了支气管肺泡灌洗.比较了CAPA患者和仅COVID-19患者的支气管肺泡灌洗NET浓度，并结合了支气管肺泡灌洗中免疫介质的现有数据和90天死亡率。
结果：我们对39例患者的43个样本进行了支气管肺泡灌洗的scRNA-seq，其中36例患者(30例男性和6例女性;14例CAPA患者)被纳入下游分析.我们对59例患者（男性46例，女性13例）进行了支气管肺泡灌洗NET分析，其中26人患有CAPA。通过scRNA-seq，CAPA患者的中性粒细胞分数显著低于单纯COVID-19患者(16%vs33%;p=0·0020).CAPA患者的剩余中性粒细胞优先遵循杂交成熟轨迹，其特征是与抗原呈递相关的基因表达。抗真菌效应子途径的转录增强。CAPA患者还显示粘膜相关的不变T细胞的消耗，T辅助细胞1和T辅助细胞17分化减少，以及巨噬细胞和单核细胞抗真菌免疫特定方面的转录缺陷。我们观察到，与仅COVID-19患者相比，CAPA患者的NETs形成增加（与瓜氨酸化组蛋白H3复合的DNA中位数为15898ng/mL[IQR4588-86419]对7062ng/mL[775-14088]；p=0·042），从而解释了通过scRNA-seq减少的中性粒细胞分数。低支气管肺泡灌洗NET浓度与CAPA患者90天死亡率增加相关。
结论：单核细胞的定性和定量紊乱，巨噬细胞,B细胞,T细胞群可能使患有严重COVID-19的患者易患CAPA。混合中性粒细胞对CAPA形成专门的反应，和适当的中性粒细胞对CAPA的反应是这些患者生存的主要决定因素。因此,检测支气管肺泡灌洗NETs对CAPA患者具有诊断和预后价值。临床医生在使用可能抑制NETosis的免疫调节疗法治疗重症COVID-19患者时应警惕曲霉病。
背景：佛兰德斯研究基金会，KULeuven,UZLeuven,VIB,基金会，欧洲区域发展基金，laCaixa基金会，佛兰德政府,地平线2020
