Pathophysiological Mechanisms of Liver Injury in COVID-19

Alexander D. Nardo; Mathias Schneeweiss-Gleixner; May Bakail; Emmanuel D. Dixon; Sigurd F. Lax; Michael Trauner

Disclosures

Liver International. 2021;41(1):20-32. 

In This Article

Potential Molecular Mechanisms for SARS-CoV-2 Tropism of the Liver

The presence of SARS-CoV-2 viral RNA has recently been demonstrated by qRT-PCR in liver among various other organs outside the respiratory tract,[52] although the exact cellular site of replication remained unspecified since nucleic acids have been isolated by whole-tissue homogenization. However, in situ hybridization analyses revealed SARS-CoV-2 virions in vessel lumens and endothelial cells of portal veins of COVID-19 liver specimens.[51] Moreover, electron microscopic analyses on liver samples from two deceased COVID-19 patients with elevated liver enzymes demonstrated the presence of intact viral particles in the cytoplasm of hepatocytes.[40]

Given recent, although still limited, discoveries,[40,51,52] hepatic tropism for SARS-CoV-2 and direct cytopathic effects should be considered as potential mechanism of COVID-19 associated liver injury, although a classic hepatitic picture has not been reported.[40,46,49–51] The availability of viral receptors at the host cell surface is a major determinant of viral tropism for a specific tissue.[53] As such, SARS-CoV-2 cell entry is mediated by the S protein of the virus, which specifically interacts with host ACE2 and TMPRSS2 (Figure 1). In order to understand whether SARS-CoV-2 might be able to infect liver cells, we explored the expression pattern of the human ACE2 and TMPRSS2 proteins using the Human Protein Atlas (data available at https://www.proteinatlas.org/ENSG00000130234-ACE2/tissue and https://www.proteinatlas.org/ENSG00000184012-TMPRSS2/tissue). Interestingly, the expression levels of the two proteins is highest in intestine and gall bladder, but it appears to be virtually absent in the liver. These data might be incomplete or lack sensitivity, since in the Human Protein Atlas ACE2 expression also seems to be absent in the lungs, where infection is definitely known to occur. In a recent study, Chai and colleagues applied single-cell RNAseq to healthy human liver samples and found that ACE2 expression levels in bile duct epithelium (cholangiocytes) is comparable to that of alveolar cells in the lungs, whereas hepatocellular ACE2 expression is low but still detectable.[54] Further confirmation of significant ACE2 and TMPRSS2 expression in liver parenchymal cells comes from bio-informatics analyses from the single-cell transcriptome database Single Cell Portal.[55] Interestingly, sinusoidal endothelial cells appear to be ACE2-negative, in line with previous observations.[56] This finding may be important considering recent reports on endothelitis of large intrahepatic vessels caused by SARS-CoV-2[48,57] and high ACE2 expression in other endothelia, including central and portal veins, which also can become infected by the virus.[51]

Of note, studies in both mice and humans revealed increased hepatic ACE2 expression in hepatocytes upon liver fibrotic/cirrhotic conditions[58,59] (and our own unpublished observations). This finding may be of great relevance since pre-existing liver injury could thereby exacerbate SARS-CoV-2 hepatic tropism. Moreover, hypoxia, which is a typical feature in severe COVID-19 cases, has been shown to be a main regulator of hepatocellular ACE2 expression.[58] This might explain why extra-pulmonary SARS-CoV-2 dissemination is mainly observed in patients manifesting ARDS and other hypoxic conditions. Importantly, inflammatory conditions/diseases in the liver, as shown for other organs,[60,61] could also upregulate ACE2 expression. Since drug-induced liver injury (DILI) may contribute to liver injury in COVID-19 patients,[62] it might be of interest to explore whether DILI or certain drugs induce hepatic ACE2 over-expression.

In vitro experiments also showed that the S protein of lineage B beta-coronaviruses significantly increases the affinity for its receptor when it is pre-incubated with trypsin, that is when it is proteolytically activated.[63] Since liver epithelial cells express trypsin[64] and a plethora of other serine-proteases which constantly remodel the extracellular matrix,[65] ACE2 expression required for SARS-CoV-2 target and recognition in the liver might be lower than in other tissues with reduced extracellular proteolytic activity.[66] In line with these findings, it has been recently discovered that the S protein of SARS-CoV-2 bears a furin-like proteolytic site never observed before in other coronaviruses of the same lineage.[67] Interestingly, furin is predominantly expressed in organs that have been proposed as permissive for SARS-CoV-2 infection, such as salivary glands, kidney, pancreas (data for The Human Protein Atlas, available at https://www.proteinatlas.org/ENSG00000140564-FURIN/tissue) and the liver.[55]

Finally, other factors, as for example ganglioside (GM1),[68] might influence S protein-ACE2 interaction. Therefore, research should also explore more deeply the S protein-ACE2 interactome to achieve new molecular and therapeutic insights.

In a recent report, Ou and colleagues tested pseudovirions containing the SARS-CoV-2 S protein for their ability to infect different cell lines. Interestingly, HuH7 cells, a hepatocyte cell line, as well as Calu3 cells, a human lung carcinoma cell line, were more efficiently transfected by viral vectors carrying the SARS-CoV-2 S protein than control pseudovirions.[69] Moreover, these studies revealed that viral entry might depend on the PIKfyve-TCP2 endocytotic pathway. A crosscheck in the Human Protein Atlas revealed that both PIKfyve and TPC2 are expressed in liver and gall bladder at comparable levels as in the lung (data available at https://www.proteinatlas.org/ENSG00000115020-PIKFYVE/tissue and https://www.proteinatlas.org/ENSG00000162341-TPCN2/tissue), highlighting the potential relevance of this pathway for hepatic tropism, which therefore expands from simple targeting and recognition to support of intracellular viral replication.

In an effort to establish a new and effective functional viromics screening approach aimed at predicting the likelihood of zoonotic events of the known lineage B betacoronaviruses, Letko and colleagues took advantage of HuH7 cells as a permissive model for SARS-CoV and SARS-CoV-2 binding and recognition,[63] further proving SARS-CoV-2 tropism for hepatocytes. Of note, HuH7 cells were described as the third most permissive cell line in this study after pulmonary (Calu3) and intestinal (CaCo2) cell models,[63] the latter representing organs with histopathologically proven SARS-CoV-2 infection. However, the ability of binding and internalizing viral particles does not necessarily imply that the cell type under investigation is also permissive for effective viral replication. In this regard, both Chu and colleagues and Harcourt et al demonstrated that HuH7 cells support SARS-CoV-2 viral replication.[70,71] Hepatocyte cell lines are now such an established permissive cell type for SARS-CoV and SARS-CoV-2 infection that HuH7 cells have also been recently used as positive control in SARS-CoV-2 immunostainings.[72]

Although the above-reported observations define hepatocytes as putative hosts for SARS-CoV-2, it is important to point out that all the data arise from studies in which cancer cell lines have been used. In order to clarify the translational potential of these observations, ACE2 protein expression in HuH7 cells should be compared with that of primary human hepatocytes. Furthermore, future investigations are needed to uncover the molecular changes induced in hepatocytes upon SARS-CoV-2 infection.

A reliable source of information comes from recent work by Yang and colleagues, who demonstrated SARS-CoV-2 tropism for hepatocytes using organoids obtained from human pluripotent stem cell (hPSC)-derived hepatocyte and primary adult human hepatocytes.[73] In these systems, pseudovirions expressing SARS-Cov-2 S protein were able to infect human hepatocytes, while SARS-CoV-2 infection resulted in robust viral replication.[73] Gene expression analyses also showed that SARS-CoV-2-infected primary hepatocytes over-express pro-inflammatory cytokines, while downregulating key metabolic processes, as reflected by the inhibition of CYP7A1, CYP2A6, CYP1A2 and CYP2D6 expression.[73]

Finally, Wang and colleagues applied electron microscopy imaging to liver samples of two deceased COVID-19 patients, and identified viral structures in hepatocytes which distinctively resemble SARS-CoV-2 virions.[40] This raises the possibility that the histopathological alterations seen in these patients may be caused by direct cytopathic effects of SARS-CoV-2[40]although a typical hepatitis pattern appears to be lacking.[40,46,49–51] However, further studies with larger biopsy/autopsy cohorts and the combined imaging (including immune electron microscopy) may be necessary to confirm these preliminary observations of hepatocellular SARS-CoV-2 presence.

Bile duct epithelial cells (cholangiocytes) participate in bile production and flow as well in immune response.[74] Single-cell sequencing of human long-term liver ductal organoid cultures showed preservation of ACE2 and TMPRSS2 expression.[75] Following SARS-CoV-2 infection, cholangiocytes underwent syncytia formation and the amount of SARS-CoV-2 genomic RNA was dramatically increased 24 hours post-infection. Similar results have been obtained when infecting adult human cholangiocyte organoids with SARS-CoV-2.[73] These observations indicate that human liver ductal organoids may be susceptible to SARS-CoV-2 infection in vitro and suggest that viral replication could also occur within the bile duct epithelium in vivo. However, despite significantly higher ACE2 expression when compared with hepatocytes, no direct evidence of SARS-CoV-2 cholangiocellular infection has been reported so far in COVID-19 patients. Since bile is primarily produced by hepatocytes and cholangiocytes, and given the continuous and direct contact between biliary fluids and the cholangiocellular apical membrane, identification of SARS-CoV-2 viral RNA or proteins in bile could be an indirect proof of SARS-CoV-2 cholangiocellular infection. At the moment, only one case report has shown SARS-CoV-2 RNA in bile,[76] whereas bile from two other small sample series tested negative.[24,49] These discrepancies might rely on the fact that the positive-tested bile sample has been obtained during surgical resolution of bile duct obstruction,[76] whereas the negatively tested bile was obtained from 48h post-mortem autopsies.[24,49]

Tight junctions allow cholangiocytes to act as a protective barrier for parenchymal liver cells from toxic bile components. Viral infection with SARS-CoV-2 decreased mRNA expression of cholangiocellular tight junction proteins such as claudin 1 in vitro,[75] implicating reduced barrier function of cholangiocytes. This in turn could cause liver injury through leakage of potentially toxic bile into the periductal space and adjacent liver parenchyma. Of note, expression of the bile acid transporters SLC10A2/ASBT and chloride channel ABCC7/CFTR was significantly down-regulated by SARS-CoV-2 infection.[75] The negative regulation of these hepatobiliary transporters may impair bile acid sensing/signalling by cholangiocytes and bicarbonate secretion, eventually contributing to biliary changes observed in COVID-19 infection.[49] Furthermore, cholangiocytes infected with SARS-CoV-2 virus upregulated inflammatory pathways, depicting the induction of a reactive cholangiocyte phenotype.[73] Future studies will have to explore whether and how SARS-CoV-2 may alter secretion of pro-inflammatory and pro-fibrogenic cytokines and contribute to the 'reactive cholangiocyte phenotype', which could propagate inflammation and fibrosis.[74]

Pre-existing chronic liver diseases seem to be independent risk factors for poor outcome in COVID-19, and cirrhosis grade has been defined as a predictor of mortality in SARS-CoV-2 infected patients[77] (Figure 2). Activation of hepatic stellate cells plays a paramount role in the progression of chronic liver disease as the main cellular source of fibrosis[78] and is induced by pro-inflammatory and pro-fibrotic cues, such as Angiotensin II, generated by the catalytic action of ACE as part of the pro-fibrotic branch of the renin-angiotensin system.[79] Of note, ACE2 counteracts ACE function by producing the anti-inflammatory and anti-fibrotic Angiotensin-(1–7) and thereby decreasing the Angiotensin II/Angiotensin-(1–7) ratio.[79] However, ACE2 expression has neither been detected in quiescent, nor in fibrogenic/activated hepatic stellate cells.[58,80–83] These findings suggest that these cells may be a rather non-permissive host for SARS-CoV-2. Nevertheless, the pro-inflammatory milieu generated by direct or indirect COVID-19-associated hepatocellular and cholangiocellular injury may pave the way for activation of hepatic stellate cells and consequent induction of fibrosis. This possibility may be even more relevant in patients with underlying CLD, such as NAFLD. Although available data suggest that COVID-19-related liver injury is mild and transitory, long-term follow-up studies will be necessary to exclude hepatic fibrosis as a potential long-term consequence of COVID-19, especially in the presence of pre-existing liver diseases.

Monocyte-derived macrophages (MoM) and alveolar macrophages are known to express ACE2,[84,85] and there is evidence of alveolar macrophage infection by SARS-CoV[85] and SARS-CoV-2 with detection of viral protein by immunohistochemistry.[24,86] However, a histopathologic assessment of ACE2 tissue distribution showed no staining in Kupffer cells and other hepatic immune cells,[56] although Kupffer cell proliferation is typically observed in livers of COVID-19 diseased.[40,49] The recent COVID-19 pandemic further prompted more in-detail investigations on ACE2 expression and de novo single-cell RNAseq analyses,[54] as also in silico evaluations of RNAseq databases[87,88] proved that Kupffer cells do not express ACE2. It has to be kept in mind, however, that all the described evidences refer to healthy human liver samples. Therefore, quantification of ACE2 expression in samples obtained from patients with underlying chronic liver disease or acute liver injury may be needed to obtain definitive insights into macrophage ACE2 expression patterns.

Of note, upon liver injury and/or Kupffer cell depletion, MoM can invade the liver and efficiently replenish the hepatic resident macrophage population[89–91] (and reviewed in detail in[92]). Although in vitro observations proved that MoM does not support efficient replication of SARS-CoV (and most probably also SARS-CoV-2), infected MoM could act as carriers of the pathogen, favouring infection of the ACE2-expressing cells in the invaded organ.[93] Furthermore, Kupffer cell activation and proliferation are frequently observed as a consequence of systemic inflammation and Kupffer cell activation has been reported in the liver specimen of deceased COVID-19 patients.[40,49] Thus, although Kupffer cells do not express ACE2, monocytic cells might play a key role in SARS-CoV-2-mediated liver injury by propagation of inflammatory stimuli.

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