Single-nucleus RNA sequencing and spatial transcriptome atlas of COVID-19 livers

By Tarun Sai LomteNov 1 2022Reviewed by Aimee Molineux

In a recent study posted to the bioRxiv* preprint server, researchers performed single-nucleus RNA sequencing (snRNA-seq) and spatial transcriptomic profiling of livers from deceased coronavirus disease 2019 (COVID-19) patients.

*Important notice: bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Background

COVID-19 presents a broad phenotypic spectrum with the potential involvement of multiple organs during the acute phase. There are limited studies on livers from COVID-19 patients impeding detailed analysis of associated liver injuries and possible long-term effects. Previously, the authors created a COVID-19 cell atlas of multiple tissues from deceased patients using snRNA-seq.

Although the lung pathobiology in COVID-19 has been investigated in depth, the impact on other organs, including the liver, has not been explored elaborately. Several factors, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, systemic inflammation, hypoxia, and drug-induced injury, may underlie the COVID-19 liver phenotype.

The study and findings

In the present study, researchers created a COVID-19 liver atlas of snRNA-seq profiles from liver specimens of 17 COVID-19 decedents. The authors used snRNA-seq to obtain 80,808 profiles from the autopsied patients, which were integrated with those from four healthy controls. These profiles were segregated into five compartments – hepatocytes, immune/blood, endothelial, biliary epithelial (BECs), and mesenchymal cells.

A spatial transcriptome atlas was generated from 62 regions of interest (ROIs) from three lobular zones and portal triad across four autopsied patients using the digital spatial profiling whole transcriptome atlas (WTA) platform. The expression of more than 18,000 genes was captured on the WTA. The portal triad and the three lobular zones had distinct expression profiles.

The lobular zone 1 had a high transcriptional activity for urea cycle, glutathione and lipid metabolism, lipoprotein assembly, steroid and fatty acid biosynthesis, and urea cycle. Zone 2 had comparable patterns but showed increased fucose biosynthesis and triglyceride catabolism. Contrastingly, zone 3 had higher drug catabolism activities.

In COVID-19 specimens, there was hepatocyte proliferation in lobular zone 1 and stress or hypoxic response pathways in lobular zone 3, which are not documented in healthy livers. Zone 3 had the highest signals of inflammation – inflammasome activation, interleukin (IL) signaling, cytokine responses, interferon (IFN)-γ binding, and apoptotic processes, which may be linked to SARS-CoV-2 infection and may not be as pronounced in healthy livers.

Hepatocytes represented the dominant compartment (63.8%) in the atlas and were divided into seven subsets (HEP1 to HEP7). HEP2 cells showed high expression of genes encoding circulating proteins (coagulation factors, albumin, apolipoproteins). HEP6 and HEP7 cells had profiles similar to those of HEP2 cells but with elevated expression of cellular senescence or apoptotic pathways in HEP6 and acute phase proteins in HEP7.

By contrast, HEP1, HEP4, and HEP3 cells had higher expression of cell differentiation, signal transduction, and wound healing pathways but exhibited decreased liver metabolic/synthetic function gene expression. Trajectory analysis from COVID-19 and healthy livers suggested a differentiation path from HEP3 cells to highly-differentiated HEP3 cells through HEP1, HEP4, and HEP5 intermediates, with HEP6 and HEP7 being the direct descendants of the HEP2 subset.

Healthy and COVID-19 cellular landscapes indicated restructuring of the COVID-19 hepatocyte compartment, the emergence of COVID-19-specific HEP7 cluster from HEP2 cells, and the expression of acute phase proteins. Next, they analyzed cell type- and donor-specific distributions of SARS-CoV-2 reads.

Hepatocytes were highly enriched with SARS-CoV-2 RNA⁺ nuclei. Viral RNA levels correlated with the expression of numerous heat shock proteins, suggesting the activation of unfolded protein response due to cellular stress. HEP4 profiles indicated activation of inflammatory reactions, concordant with the SARS-CoV-2 infection of epithelial cells.

BECs expressed lineage markers and spanned a broad spectrum with six subsets (BEC1 to BEC6). BEC4, BEC5, and BEC6 subsets showed distinct profiles consistent with reactive cholangiocytes or pro-fibrogenic ductular reactions in chronic liver diseases. BEC1, BEC2, BEC5, and BEC6 subsets increased, while BEC3 and BEC4 subsets declined in COVID-19 livers relative to healthy controls.

The blood/immune cell compartment of COVID-19 livers comprised T cells, natural killer (NK) cells, B cells, mast cells, and monocytes/macrophages/Kupffer cells (KCs). Compared to healthy livers, there was an extensive restructuring of myeloid and T cell compartments in COVID-19 livers. Naïve CD8⁺ T cells were significantly reduced, whereas cytotoxic effector/memory T cells and apoptotic naïve T-cell-like populations were enriched substantially in COVID-19 livers.

No differences were observed in classical/inflammatory KCs in COVID-19 livers. No macrophage subset showed elevated expression of chemokine receptors, indicating deficient infiltration of monocyte-derived macrophages that reflected a degree of pulmonary tropism or immune exhaustion. The endothelial cell compartment, which spanned across 12 subsets, was highly impacted in COVID-19 livers relative to controls.

Mesenchymal cells, across eight subsets, represented all major cell lineages in the liver. The authors observed fibrogenic activation in the mesenchymal subsets in COVID-19 livers. Cell-cell communication analysis of COVID-19 snRNA-seq data showed a multi-cell interaction hub involving hepatocytes, endothelial cells, and mesenchymal cells. These analyses revealed a diverse source of fibrogenic activation in COVID-19.

Further validation of the pro-fibrotic phenotype of COVID-19 livers was provided through a liver histopathologic survey of four donors. Sinusoidal fibrosis and stellate cell activation were strikingly common in the four donors. In addition, three patients exhibited moderate to extensive ductular reaction or cholangiocyte proliferation.

Conclusions

The researchers observed extensive remodeling of cellular and expression landscapes in COVID-19 livers, suggesting ductular reaction, hepatocellular injury, fibrogenesis, and neo-vascular expansion. The hepatocytes with SARS-CoV-2 RNA had an increased expression of pro-inflammatory and acute phase proteins. There were massive changes in the composition and expression profiles of non-parenchymal cells in COVID-19 livers.

The observed pattern of fibrosis could not be explained by an underlying chronic liver disease alone and may have been caused by localized and systemic sepsis-like effects of COVID-19. The COVID-19-induced expression/cellular changes indicate a sub-clinical but profound impact of the disease on the liver, despite the absence of a significant liver injury, and may have long-term implications on the health of COVID-19 survivors.

*Important notice: bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.