High-dimensional comparison of monocytes and T cells in post-COVID and idiopathic pulmonary fibrosis

Up to 30% of hospitalized COVID-19 patients experience persistent sequelae, including pulmonary fibrosis (PF). We examined COVID-19 survivors with impaired lung function and imaging worrisome for developing PF and found within six months, symptoms, restriction and PF improved in some (Early-Resolving COVID-PF), but persisted in others (Late-Resolving COVID-PF). To evaluate immune mechanisms associated with recovery versus persistent PF, we performed single-cell RNA-sequencing and multiplex immunostaining on peripheral blood mononuclear cells from patients with Early- and Late-Resolving COVID-PF and compared them to age-matched controls without respiratory disease. Our analysis showed circulating monocytes were significantly reduced in Late-Resolving COVID-PF patients compared to Early-Resolving COVID-PF and non-diseased controls. Monocyte abundance correlated with pulmonary function forced vital capacity and diffusion capacity. Differential expression analysis revealed MHC-II class molecules were upregulated on the CD8 T cells of Late-Resolving COVID-PF patients but downregulated in monocytes. To determine whether these immune signatures resembled other interstitial lung diseases, we analyzed samples from Idiopathic Pulmonary Fibrosis (IPF) patients. IPF patients had a similar marked decrease in monocyte HLA-DR protein expression compared to Late-Resolving COVID-PF patients. Our findings indicate decreased circulating monocytes is associates with decreased lung function and uniquely distinguishes Late-Resolving COVID-PF from Early-Resolving COVID-PF, IPF, and non-diseased controls. Overall design: Patients with IPF donated peripheral blood mononuclear cells (PBMCs) in the outpatient setting while in a stable clinical state. Three control PBMC samples from age-matched patients with no known pulmonary disease were prepared and sequenced at the Mayo Clinic (IRB #:19-012187). We recruited a subset of patients from the University of Virginia COVID-19 survivor clinic (IRB #:13166) with restrictive lung physiology on pulmonary function tests (PFTs) and features consistent with pulmonary fibrosis on chest CT performed at the associated visit. Radiographic features indicative of possible development of pulmonary fibrosis included bilateral reticulation, traction bronchiectasis, and/or honeycomb change in peripheral and basilar distribution, similar to the presently recognized progressive pulmonary fibrosis clinical radiologic phenotype, and similar to previously defined COVID-19 pulmonary fibrosis characteristics. PFT and chest imaging was performed in association with the patient's first visit to the outpatient COVID-19 survivor clinic. Early- and Late-Resolvers were identified by comparing chest imaging and PFT values from the patient's first and subsequent visit. Patients with COVID-19 associated pulmonary fibrosis (COVID PF) were followed for 6 months or until the patient clinically improved. COVID PF patients were age-matched to IPF patients to control for age-related differences in peripheral immune signatures.

高达30%的住院新型冠状病毒肺炎（COVID-19）患者会出现持续性后遗症，其中包括肺纤维化（pulmonary fibrosis, PF）。我们针对肺功能受损、影像学提示存在肺纤维化患病风险的新冠康复患者开展研究，发现在发病后6个月内，部分患者的相关症状、限制性通气障碍及肺纤维化表现得到缓解（我们将其命名为早缓解型新冠相关肺纤维化（Early-Resolving COVID-PF)），而另一部分患者的上述表现则持续存在（即晚缓解型新冠相关肺纤维化（Late-Resolving COVID-PF)）。

为探究与病情缓解及持续性肺纤维化相关的免疫机制，我们分别采集早缓解型、晚缓解型新冠相关肺纤维化患者的外周血单个核细胞（peripheral blood mononuclear cells, PBMC），对其进行单细胞RNA测序与多重免疫染色，并将结果与年龄匹配的无呼吸道疾病健康对照人群进行对比分析。

我们的分析结果显示，与早缓解型新冠相关肺纤维化患者及非疾病对照相比，晚缓解型新冠相关肺纤维化患者的循环单核细胞数量显著降低。单核细胞丰度与肺功能指标用力肺活量（forced vital capacity）及弥散量（diffusion capacity）呈显著正相关。差异表达分析表明，晚缓解型新冠相关肺纤维化患者的CD8 T细胞表面MHC-II类分子表达上调，而单核细胞表面该类分子的表达则出现下调。

为明确上述免疫特征是否与其他间质性肺疾病存在相似性，我们还分析了特发性肺纤维化（Idiopathic Pulmonary Fibrosis, IPF）患者的样本。与晚缓解型新冠相关肺纤维化患者相比，特发性肺纤维化患者的单核细胞HLA-DR蛋白表达同样出现显著降低。

本研究结果表明，循环单核细胞数量减少与肺功能下降密切相关，且该特征可将晚缓解型新冠相关肺纤维化与早缓解型新冠相关肺纤维化、特发性肺纤维化及非疾病对照人群明确区分。

### 实验设计概况
特发性肺纤维化患者在临床状态稳定的门诊就诊期间捐赠了外周血单个核细胞。另有3份来自年龄匹配的无已知肺部疾病患者的外周血单个核细胞样本，由梅奥诊所（Mayo Clinic）完成制备与测序（IRB #:19-012187）。
我们从弗吉尼亚大学新冠康复患者门诊（IRB #:13166）招募了一组患者，这些患者在就诊时的肺功能测试（pulmonary function tests, PFTs）显示存在限制性通气障碍，且同期胸部CT影像学表现符合肺纤维化特征。提示可能发生肺纤维化的影像学特征包括：双侧网状影、牵引性支气管扩张，以及/或外周及基底部区域的蜂窝样改变，这与目前公认的进行性肺纤维化临床放射表型一致，也与此前定义的新冠相关肺纤维化特征相符。
肺功能测试与胸部影像学检查均在患者首次前往新冠康复患者门诊就诊时完成。通过对比患者首次就诊及后续随访的胸部影像学与肺功能测试结果，我们将患者划分为早缓解型与晚缓解型新冠相关肺纤维化患者。新冠相关肺纤维化（COVID PF）患者均接受了为期6个月的随访，直至患者临床症状改善。所有新冠相关肺纤维化患者均与特发性肺纤维化患者进行年龄匹配，以控制外周免疫特征中的年龄相关差异。