Molecular and immune signatures, and pathological trajectories of fatal COVID‐19 lungs defined by in situ spatial single‐cell transcriptome analysis

Abstract

Despite intensive studies during the last 3 years, the pathology and underlying molecular mechanism of coronavirus disease 2019 (COVID‐19) remain poorly defined. In this study, we investigated the spatial single‐cell molecular and cellular features of postmortem COVID‐19 lung tissues using in situ sequencing (ISS). We detected 10 414 863 transcripts of 221 genes in whole‐slide tissues and segmented them into 1 719 459 cells that were mapped to 18 major parenchymal and immune cell types, all of which were infected by SARS‐CoV‐2. Compared with the non‐COVID‐19 control, COVID‐19 lungs exhibited reduced alveolar cells (ACs) and increased innate and adaptive immune cells. We also identified 19 differentially expressed genes in both infected and uninfected cells across the tissues, which reflected the altered cellular compositions. Spatial analysis of local infection rates revealed regions with high infection rates that were correlated with high cell densities (HIHD). The HIHD regions expressed high levels of SARS‐CoV‐2 entry‐related factors including ACE2, FURIN, TMPRSS2 and NRP1, and co‐localized with organizing pneumonia (OP) and lymphocytic and immune infiltration, which exhibited increased ACs and fibroblasts but decreased vascular endothelial cells and epithelial cells, mirroring the tissue damage and wound healing processes. Sparse nonnegative matrix factorization (SNMF) analysis of niche features identified seven signatures that captured structure and immune niches in COVID‐19 tissues. Trajectory inference based on immune niche signatures defined two pathological routes. Trajectory A primarily progressed with increased NK cells and granulocytes, likely reflecting the complication of microbial infections. Trajectory B was marked by increased HIHD and OP, possibly accounting for the increased immune infiltration. The OP regions were marked by high numbers of fibroblasts expressing extremely high levels of COL1A1 and COL1A2. Examination of single‐cell RNA‐seq data (scRNA‐seq) from COVID‐19 lung tissues and idiopathic pulmonary fibrosis (IPF) identified similar cell populations consisting mainly of myofibroblasts. Immunofluorescence staining revealed the activation of IL6‐STAT3 and TGF‐β‐SMAD2/3 pathways in these cells, likely mediating the upregulation of COL1A1 and COL1A2 and excessive fibrosis in the lung tissues. Together, this study provides a spatial single‐cell atlas of cellular and molecular signatures of fatal COVID‐19 lungs, which reveals the complex spatial cellular heterogeneity, organization, and interactions that characterized the COVID‐19 lung pathology.

Integrative single‐cell transcriptome analysis provides new insights into post‐COVID‐19 pulmonary fibrosis and potential therapeutic targets

“…For instance, collagen gene upregulation was observed in fibroblasts in both COVID‐19 and IPF patient cohorts (Figure ). This finding is consistent with a recent study by Das et al 41 characterizing the molecular profiles of COVID‐19. The authors emphasized the crucial role of collagen genes in the pulmonary pathology resulting from COVID‐19.…”

“…Our results showed that these pathways were reversed after treatment with antifibrotic agents (Figure ). Furthermore, the expression level of COL1A1 was highly correlated with the proximity of fibroblasts to infiltrating macrophages or monocytes 41 . These results in spatial levels, support that fibroblast and infiltrated macrophages were key pathology players in PCPFs.…”

Differential effects of microRNAs miR‐21, miR‐99 and miR‐145 on lung regeneration and inflammation during recovery from influenza pneumonia

“…This suggests that global RNA‐Seq analysis could not detect expression differences of certain genes that may be differentially regulated in the proliferating AT2 cells between the two miRNA treatment groups. To resolve this, future studies should thus harness spatial single‐cell transcriptome analysis of different cell types in the lung 36–38 …”

“…To resolve this, future studies should thus harness spatial single-cell transcriptome analysis of different cell types in the lung. [36][37][38] 3.7 | Downregulation of miR-99a of influenzainfected mice impacts growth-related processes…”

Obesity fosters severe disease outcomes in a mouse model of coronavirus infection associated with transcriptomic abnormalities

“…A recent study found high levels of fibroblast markers, such as collagen type 1 alpha 1 (Col1a1) and collagen type 1 alpha 2 (Col1a2), associated with organizing pneumonia (OP) in the lungs of patients with severe COVID-19 compared to healthy individuals. 81 3.4 | Gene Set Variation Analysis (GSVA) showed significant differences between MHV-1 infected lean and obese C3H/HeJ mice We further utilized our RNAseq data to carry out Gene Set Variation Analysis (GSVA) and compared the impact of MHV-1 infection and obesity on differential enrichment of disease-relevant immune cell, inflammatory, and cellular pathway gene signatures 12,33,83 (Figure 6A,B). Several immune cell type signatures associated with COVID-19 patient disease pathology were also changed in MHV-1 infected mice, including enrichment of inflammatory neutrophils, low density granulocytes (LDGs), monocytes, and activated B cells as well as de-enrichment of T cells (Figure 6A).…”