COVID-19 single cells studies in BAL.
The majority of single-cell studies to date were performed on peripheral blood mononuclear cells (PBMC), a minority on NP swabs and BAL. Few studies have dissected the epithelial and immune profiles of BAL derived from severe COVID-19 patients at a single-cell level. Wauters et al revealed infected lung epithelial cells, a significant proportion of neutrophils and macrophages involved in viral clearance.50 They performed single-cell deep-immune profiling BAL from 5 patients with mild and 26 with critical COVID-19 (compared to non-COVID-19 pneumonia and normal lung) showing divergent immunologic profiles. In mild COVID-19, CD8+ resident-memory (TRM) and CD4+ T-helper-17 (TH17) cells undergo active expansion with good effector functions, while in critical cases they remain more naïve. Vice versa, CD4+ T-cells with T-helper-1 characteristics (TH1-like) and CD8+ T-cells expressing exhaustion markers (TEX-like) are enriched halfway their trajectories in mild COVID-19, where they also exhibit good effector functions, while in critical COVID-19 they show evidence of inflammation-associated stress. Monocyte-to-macrophage trajectories show that chronic hyperinflammatory monocytes are enriched in critical COVID-19, while alveolar macrophages, otherwise characterized by anti-inflammatory and antigen-presenting characteristics, are depleted. Moreover, in critical COVID-19, monocytes contribute to an ATP- purinergic signaling-inflammasome footprint that could enable COVID-19 associated fibrosis and worsen disease-severity.50Liao et al evaluated BAL from 3 moderate and 6 severe COVID-19 and found abundant pro-inflammatory monocytes derived macrophages in patients with severe COVID-19, whereas highly clonally expanded CD8+ T cells characterized moderate COVID-19 cases.51 Patients with severe/critical infection had much higher levels of inflammatory cytokines, particularly interleukin (IL)-8, IL-6 and IL-1β, expressed by macrophages that in severe patients may contribute to local inflammation by recruiting monocytic cells and neutrophils thought CCR1 and CXCR2, while in moderate cases can produce more T cell attracting chemokines through CXCR3 and CXCR6.51 He et al performed single-cell RNA sequencing (sc-RNA-seq) in the leukocytes and epithelial cells of 3 SARS-Cov-2 induced ARDS.52 They detected 23 cells with viral mRNA reads, but minimal number of expressed genes, thus indicating that SARS-Cov-2 suppresses host gene expression. These cells were identified as monocytes/neutrophils and club cells. Compared to healthy controls club cells showed a significantly elevated mucins genes expression (MUC5AC, MUC5BMUC4, MUC16 and MUC20). The mucin secretion seems stimulated through the innate immune regulators IL-1β and TNF-α (were found 6 transcription factors involved in IL-1β and TNF- α induced MUC5B promoter activation). Four critical surfactant proteins (SPs)— SP-A, SP-B, SP-C, and SP-D, known to maintain the structural integrity of alveoli, were down-regulated in COVID-19 disease and the level of NKX2-1, the transcription factor required for surfactant synthesis was also reduced, thus indicating the loss of alveoli integrity and the possible pathogenesis of ARDS in COVID-19.52 The transcriptomic signature of major regulators of innate immunity (monocytes, neutrophils and macrophages) in severe COVID-19 indicates different immune profiles among COVID-19 patients: Liao et al. showed abundant macrophages expressing FCN1 in BAL of COVID-19 patients, whereas He et al. noticed only a minor increase in FCN1+ macrophages, with a significant decrease in FCN1+ monocytes/neutrophils.51,52 By analyzing scRNA-seq data of BAL from 6 severe COVID-19, 3 recovered COVID-19 with mild symptoms and 10 heathy controls, Chen et al. showed high expression of SARS-Cov-2 receptor ACE2 and TMPRSS2 in club and ciliated cells of patients.53 In severe COVID-19 high neutrophils with excessive expression of cytokines were noted and the dysregulated cytokines/receptors interplay among lung epithelial cells and immune cells correlated with disease severity (ANXA1/FPR2 and TNFSF13/TNFRSF1A interactions between club and macrophage or neutrophils, CXCL2/DPP4 interaction between club and T/NK cells, and ANXA1, C3, CXCL2, SAA1, TNFSF13 expressions in lung epithelial cells).53 In conclusion scRNA-seq studies can reveal information of critical importance in the understanding of COVID-19 pathogenesis. However, current data on BAL are limited, mostly derived from small sample sizes studies and with large difficulties in validating most conclusions across datasets, possibly due to inconsistent mapping between different diseases stages and different protocols used. Therefore, conclusions from these early scRNA-seq studies of COVID-19 patients may not always be robust and need to be validated before fully relied upon.54