Expansion of profibrotic monocyte-derived alveolar macrophages in patients with persistent respiratory symptoms and radiographic abnormalities after COVID-19 [PASC]

As many as 10–30% of the nearly 750 million survivors of COVID-19 develop persistent symptoms that define the post-acute sequelae of COVID-19 (PASC) syndrome. To understand the molecular basis of this syndrome, we combined a machine learning based analysis of lung computed tomography (CT) with flow cytometry and single cell RNA-sequencing analysis of bronchoalveolar lavage fluid and nasal curettage samples in a cohort of thirty-five patients with respiratory symptoms and radiographic abnormalities more than 90 days after infection with COVID-19. CT images from patients with PASC revealed primarily fibrotic abnormalities involving 73 +/- 28% of the lung, most of which improved on subsequent imaging. The presence of profibrotic monocyte-derived alveolar macrophages in BAL fluid was significantly associated with increased levels of alveolar cytokines and fibrotic abnormalities on CT. Persistent infection with SARS-CoV-2 was identified in 6 patients and secondary bacterial or viral infections in two others. These findings suggest persistent alveolar inflammation characterized by the ongoing recruitment of monocyte derived alveolar macrophages is associated with fibrotic CT changes in some COVID-19 survivors with implications for diagnosis and therapy. Bronchoalveolar lavage (BAL) fluid from 25 patients with respiratory PASC was obtained at Northwestern Medicine Comprehensive COVID-19 Center. BAL fluid was flow-sorted to enrich for live CD45+CD15- cells and single cell RNA-seq libraries were prepared with 10x 5' gene expression kit. Nasal epithelial samples were collected from 5 patients with respiratory PASC was obtained at Northwestern Medicine Comprehensive COVID-19 Center. Briefly, patients were seated and asked to extend their neck. A nasal curette (Rhino-Pro; VWR) was inserted into either nare and gently slid in the direction of posterior to anterior ~1 cm along the lateral inferior turbinate. Five curettes were obtained per participant. The curette tip was then cut and placed in 2 ml hypothermosol and stored at 4 C until processing. A single-cell suspension was generated using the cold-active dispase protocol reported by Deprez et al. AJRCCM, 2020 with slight modification. Specifically, ethylenediaminetetraacetic acid (EDTA) was omitted and cells were dispersed by pipetting 20 times every 5 min using a 1 ml tip instead of tritration using a 21/23 G needle. The final concentration of protease from Bacillus licheniformis was 10 mg ml−1. The total digestion time was 30 min. Single cell RNA-seq libraries were prepared with 10x 5' gene expression kit. Libraries were sequenced on Illumina NovaSeq 6000. Gene expression matrices were generated with 10x Cell Ranger v7.0.0 (exon-only mode) software and reads were aligned to a custom hybrid genome containing GRCh38.98 and SARS-CoV-2 (NC_045512.2).