Single-cell landscape of bronchoalveolar immune cells in patients with COVID-19

While critical for host defense, innate immune cells are also pathologic drivers of acute respiratory distress syndrome (ARDS). Innate immune dynamics during COVID-19 ARDS, compared to ARDS from other respiratory pathogens, is unclear. Moreover, mechanisms underlying beneficial effects of dexamethasone during severe COVID-19 remain elusive. Using scRNA-seq and plasma proteomics, we discovered that compared to bacterial ARDS, COVID-19 was associated with expansion of distinct neutrophil states characterized by interferon (IFN) and prostaglandin (PG) signalling. Dexamethasone during severe COVID-19 depleted circulating neutrophils, altered IFNactive neutrophils, downregulated interferon-stimulated gene, and activated IL1R2+ve neutrophils. Dexamethasone also expanded immunosuppressive immature neutrophils and remodeled cellular interactions by changing neutrophils from information receivers into information providers. Male patients had higher proportions of IFNactive neutrophils, preferential steroid-induced immature neutrophil expansion, and possibly different effects on outcome. Our single-cell atlas (www.biernaskielab.ca/COVID_neutrophil) defines COVID-19-enriched neutrophil states and molecular mechanisms of dexamethasone action to develop targeted immunotherapies for severe COVID-19. Overall design: Leukocyte and lymphocyte preparation: For lymphocyte preparation, whole blood (2mL) was collected in 5mL polystyrene round-bottom tubes. Tubes were spun (15min, 3000rpm, Room Temperature [RT]) and plasma was removed. 100 microL of Isolation Cocktail and 100 microL of Rapid Spheres (Easy SepTM Direct Human Total Lymphocytes Isolation Kit: 19655, StemCell Technologies) were added to the remaining whole blood. After mixing and 5min incubation at RT, the sample volumes of ≤ 2.5mL blood were topped up to 5mL with D-PBS+2%FBS + 1mM EDTA. The diluted sample was incubated in the magnet without lid for 5min, at RT. This last step was repeated twice before cell resuspension in 5mL of PBS+0.04% BSA. After 2 washes in PBS+0.04% BSA, and centrifugation for 5 min at 2000rpm, 7500 lymphocytes were resuspended in 25 microL of PBS+0.04% BSA. For leukocytes preparation, whole blood was collected in heparin containing vacutubes and 12 microL of 0.5M EDTA with 1mL of PBS+2% FBS and 50 microL of RBC of EasySep RBC Depletion spheres (EasySepTM RBC Depletion Reagent: 18170, Stem Cell Technologies) were added. After 5 min of magnet incubation, at RT, tubes were inverted and poured into a new tube. 50 microL of RBC depletion spheres were added. 5 min after incubation on the magnet, the tube was inverted, and cell suspension was poured into a new 15mL tube. After 2 washes in 5mL of PBS+0.04% BSA and centrifugation at 2000rpm for 5 min at 20oC, cells were resuspended in 25 microL of PBS+0.04% BSA. Single-cell RNA-Seq library construction, alignment, and quality control: A total of 15,000 single cells (containing an equal proportion of leukocytes and lymphocytes) were loaded for partitioning using 10X Genomics NextGEM Gel Bead emulsions. All samples were processed as per manufacturer’s protocol. Quality control and pre-loading cDNA quantification was performed using TapeStation D1000 ScreenTape assay. Sequencing was performed using Illumina NovaSeq S2 and SP 100 cycle dual lane flow cells over multiple rounds to ensure each sample received approximately 32,000 reads per cell. Sequencing reads were aligned using CellRanger 3.1.0 pipeline29 to two sets of reference genomes: 1. standard pre-built GRCh38 human reference, and 2. a modified GRCh38 reference with two SARS-CoV-2 genomes (MT412228 and MN908947.3) appended. Since no SARS-CoV-2 reads were detected in whole blood single-cell samples30, all analyses presented in this manuscript were performed using standard pre-built GRCh38 reference. Samples that passed alignment QC were aggregated into single datasets using CellRanger aggr with between-sample normalization to ensure each sample received an equal number of mapped reads per cell. Aggregated non-dexamethasone-treated COVID-19 (n = 12) and bacterial ARDS (n = 9) samples recovered 1,872,659 cells that were sequenced to 38,410 post-normalization reads per cell. Likewise, aggregated COVID-19 samples with (n = 9) or without (n = 12) dexamethasone recovered 1,748,551 single cells sequenced to 51,415 post-normalization reads per cell. Processed data availability: Single-cell datasets can be further explored on our companion portal at http://biernaskielab.ca/COVID_neutrophil or http://biernaskielab.com/COVID_neutrophil. Velocyto-generated LOOM files and processed R objects are available for reanalysis from: http://doi.org/10.6084/m9.figshare.14330795. Whole blood bulk RNA-Seq datasets employed as an independent validation cohort were downloaded from GSE157103. BALF scRNA-Seq datasets from severe and moderate COVID-19 were downloaded from GSE145926. Processed BALF scRNA-Seq objects from patients with bacterial pneumonia and COVID-19 (archived at GSE167118) were downloaded from authors’ archive: https://figshare.com/articles/dataset/_/13608734. Mass spectrometry datasets are available via ProteomeXchange Consortium via the PRIDE partner repository with identifier PXD028429.

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