key: cord-0794085-yklx34kp
authors: De Smet, Vincent; Verhulst, Stefaan; van Grunsven, Leo A.
title: Single cell RNA sequencing analysis did not predict hepatocyte infection by SARS-CoV-2
date: 2020-05-28
journal: J Hepatol
DOI: 10.1016/j.jhep.2020.05.030
sha: bab9f628d9c3b34c9a80b6aa50f38f3e6706a573
doc_id: 794085
cord_uid: yklx34kp

nan

Single cell RNA sequencing analysis did not predict hepatocyte infection by SARS-CoV-2 arguments for viral replication and cytopathy in infected hepatocytes which provides a partial explanation for the high prevalence of abnormal liver laboratory tests in COVID-19 patients 1 .

The hypothesis of a direct liver cell infection by SARS-CoV-2 was already opted in the past, including the last EASL-ESCMID position paper 2 . It certainly seemed that a risk stratification for direct liver cell infection could be predicted based on single cell transcriptomic data for the SARS-CoV-2 entry factors ACE2 (SARS-CoV-2 cellular entry) and TMPRSS2 (spike protein priming) 3 . In an endeavor to provide this risk stratification, many researchers reported on the analysis of single cell RNA sequencing (scRNASeq) datasets. The general conclusion was that the liver is a low-risk organ for SARS-CoV-2 infection 4 , though cholangiocytes proved to be the cells with the highest ACE2 expression 5, 6 . This led to the hypothesis that cholangiocytes are the most likely target of a direct SARS-CoV-2 infection in the liver 2 .

We did note, however, that these scRNASeq analysis manuscripts analyzed only one liver dataset, did not focus on liver tissue specifically or reported only on ACE2 expression without considering TMPRSS2 expression. Additionally, since substantial clinical data on COVID19 infection and chronic liver disease (CLD) is currently limited, we thought it would be informative to evaluate the potential vulnerability of individual cell types in CLD patients.

An enhanced ACE2 and TMPRSS2 expression in cirrhotic livers would leave these patients potentially more susceptible to liver infection with possible worse disease progression.

To address these issues, we analyzed three publicly available human liver datasets published by Aizarani et al 7 Fig. 1-2) . Hepatocytes from the datasets by Aizarani In conclusion, scRNAseq analyis does not point towards hepatocytes as a likely point of entry for SARS-CoV-2 infection. The low expression of ACE2 seen in this data presumably represents technical limitations of the scRNAseq technique, rather than an absolute absence of ACE2 in these cells leading to an underestimation of ACE2 expressing hepatocytes.

Indeed, even in alveolar epithelial type II cells, the cell type playing a crucial role in SARS-CoV pathogenesis, ACE2 expression levels were reported to be low in single cell analysis 6 .

Interestingly, while the percentage of ACE-TMPRSS2 co-expressing hepatocytes is extremely low in the two datasets containing representative hepatocytes 8, 7 , it is not zero. Since the human liver is estimated to contain tens of billions of hepatocytes 10 , a data set including cirrhotic livers from patients undergoing orthotopic liver transplantation, caused by non-alcoholic liver disease, alcoholic liver disease and Primary biliary cholangitis. All raw cell counts were normalized, scaled and clustered using principle component analysis. Cell types were identified using the same markers as in the original papers and general markers for immune cells (KLRF1, FCN1, PTPRC), endothelia (CLEC4G, PECAM1), mesenchymal cells (TIMP1, COL1A1, ACTA2), hepatocytes (TTR, ALB) and cholangiocytes (KRT7, EPCAM) were controlled in each dataset. The general markers are visualized using dot plots for each dataset where the size of the circle represents proportion of cells within the population and the color gradient represents the expression level of the gene from grey (low) to blue (high). Figure 2 . Gene expression levels of ACE2 and TMPRSS2 in liver cell populations. TSNE or UMAP displays of scRNA sequencing data from healthy and cirrhotic human livers. ACE2 and TMPRSS2 expression levels are visualized on TSNE or UMAP plots by a color gradient from grey (low) to blue (high). Note that the hepatocytes in Ramachandran dataset plots are attached to the cholangiocyte population, indicating that these cells undergo a ductular reaction. This is confirmed in Supplemental Figure 1 where these "small hepatocytes" are also positive for KRT7 and EPCAM.

SARS-CoV-2 infection of the liver directly contributes to hepatic impairment in patients with COVID-19

Care of patients with liver disease during the COVID-19 pandemic: EASL-ESCMID position paper

SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor

Single-cell RNA-seq data analysis on the receptor ACE2 expression reveals the potential risk of different human organs vulnerable to 2019-nCoV infection

Specific ACE2 Expression in Cholangiocytes May Cause Liver Damage After 2019-nCoV Infection

SARS-CoV-2 entry factors are highly expressed in nasal epithelial cells together with innate immune genes

A human liver cell atlas reveals heterogeneity and epithelial progenitors

Single cell RNA sequencing of human liver reveals distinct intrahepatic macrophage populations

Resolving the fibrotic niche of human liver cirrhosis at single-cell level

An estimation of the number of cells in the human body