Molecular Profiling of Coronavirus Disease 2019 (COVID-19) Autopsies Uncovers Novel Disease Mechanisms

Abstract

Current understanding of COVID-19 pathophysiology is limited by disease heterogeneity, complexity, and a paucity of studies assessing patient tissues with advanced molecular tools. Rapid autopsy tissues were evaluated using multi-scale RNASeq methods (bulk, single-nuclei, and spatial RNASeq next-generation sequencing) to provide unprecedented molecular resolution of COVID-19 induced damage. Comparison of infected/uninfected tissues revealed four major regulatory pathways. Effectors within these pathways could constitute novel therapeutic targets, including the complement receptor C3AR1 , calcitonin receptor like receptor or decorin . Single-nuclei RNA sequencing of olfactory bulb and prefrontal cortex highlighted remarkable diversity of coronavirus receptors. ACE2 was rarely expressed, while BSG showed diffuse expression, and ANPEP was associated with vascular/mesenchymal cell types. Comparison of lung and lymph node tissues from patients with different symptomatology (one died after a month-long hospitalization with multi-organ involvement, the other after a few days of respiratory symptoms) with digital spatial profiling resulted in distinct molecular phenotypes. Evaluation of COVID-19 rapid autopsy tissues with advanced molecular techniques can identify pathways and effectors, map diverse receptors at the single-cell level, and help dissect differences driving diverging clinical courses among individual patients. Extension of this approach to larger datasets will substantially advance the understanding of the mechanisms behind COVID-19 pathophysiology.

Molecular analysis of vascular gene expression

“…[80][81][82][83] All these studies aim to generate a platform that can eventually be used to evaluate disease-induced changes in a systematic way, thereby providing molecular insights and identifying potential targets for therapeutic interventions, as previously illustrated for COVID-19. [73][74][75] As these projects are ongoing, current work is directed at compiling curated scRNASeq data sets to provide scientists the opportunity to explore data from published scRNA-Seq studies and perform their own analyses. Examples include PanglaoDB, 84 which contains human and mouse data from a broad range of cells, and EndoDB, a database specifically focusing on the murine endothelium.…”

“…72 Furthermore, scRNASeq is being used to gain insights into the cellular and molecular mechanisms contributing to COVID-19 pathogenesis, demonstrating differences in (endothelial) cell composition and gene expression profiles in the lung, as well as in other organs. [73][74][75] Whereas these examples already indicate the power of scRNA-Seq approaches, several potential limitations should be noted in tissue, enrichment strategies via FACS can be considered to make it more cost effective. However, this can be especially difficult for ECs, since there is no "one size fits all" protocol for this enrichment, as the embedding of ECs in different microenvironments requires optimized dissociation conditions to preserve sample integrity and avoid biases due to under-or overdigestion of the tissue sample.…”

“…While efforts by large consortia are using scRNASeq to profile and categorize every cell in the body, 77‐79 previous studies focused on generating cell atlases of particular organs or (endothelial) cell types 80‐83 . All these studies aim to generate a platform that can eventually be used to evaluate disease‐induced changes in a systematic way, thereby providing molecular insights and identifying potential targets for therapeutic interventions, as previously illustrated for COVID‐19 73‐75 . As these projects are ongoing, current work is directed at compiling curated scRNASeq data sets to provide scientists the opportunity to explore data from published scRNASeq studies and perform their own analyses.…”

“…For example, one of the first studies using scRNAseq to further evaluate vascular heterogeneity in the brain identified the transcriptional basis for the gradual changes in phenotypes along the arteriovenous axis, 71 and more recently it was shown that the alveolar endothelium consists of two distinct EC subtypes with each expressing different pro‐ and anticoagulant factors 72 . Furthermore, scRNASeq is being used to gain insights into the cellular and molecular mechanisms contributing to COVID‐19 pathogenesis, demonstrating differences in (endothelial) cell composition and gene expression profiles in the lung, as well as in other organs 73‐75 …”

Genomic surveillance of SARS-CoV-2 in patients presenting neurological manifestations

“…For example, neuroinvasion and neurovirulence of the human coronavirus OC43 (HCoV-OC43), SARS-CoV-1 and Middle East Respiratory Syndrome (MERS)-CoV were reported in transgenic mice [ 20 , 21 ]. Importantly, ACE2 was shown to be highly expressed in neurons, astrocytes, and oligodendrocytes [ 22 , 23 ]. The SARS-CoV-2 ability to infect the human brain has been further analyzed in human neural progenitor cells and brain organoids [ 24 , 25 ].…”