HEPATIC INJURY IN COVID-19

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In patients with COVID-19, the most commonly used indicators of liver function impairment are liver transaminase, bilirubin, and albumin levels. Abnormal levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and total bilirubin were reported in 11%-56.3%, 15.0%-86.8%, and 2.7%-30.6% of patients with COVID-19, respectively, whereas 2%-11% of such patients had pre-existing liver disease[3-8].

In a recent study involving 228 patients with COVID-19, who did not have chronic liver disease (CLD), abnormal liver function was observed in 129 (56.3%) patients, which included elevations in the levels of ALT [84 (36.8%)], AST [58 (25.4%)], total bilirubin [59 (25.9%)], and gamma-glutamyl transferase [67 (29.5%)][9].

In a study on 99 patients, Chen et al[10] reported elevated levels of ALT and AST in 28 (28%) and 35 (35%) patients, respectively, and hypoalbuminemia and hyperbilirubinemia in 97 (98%) and 18 (18%) patients, respectively. In addition, several studies revealed that liver damage is more prevalent in severe cases of COVID-19 than in mild cases.

In a large sample multicenter study[11], abnormally elevated levels of ALT were observed in 28.1% of critically ill patients and in 19.8% of non-critically ill patients, and of AST in 18.2% of non-critically ill patients and 39.4% of critically ill patients. Huang et al[4] also indicated that patients admitted to the intensive care unit (ICU) not only had higher plasma levels of the inflammatory indices [interleukin (IL)-2, IL-7, IL-10, tumor necrosis factor (TNF)-α, etc.], but also had abnormally high levels of AST [8 (62%) of 13 patients] compared with non-ICU patients [7 (25%) of 28 patients].

A recent descriptive study confirmed that the levels of ALT (35 vs 23, normal range 9-50 U/L, P = 0.007) and AST (52 vs 29, normal range 5-21 U/L, P < 0.001) were significantly higher in ICU patients[12]. In a Japanese cohort study concerning COVID-19, patients were classified into mild, moderate, and severe groups, respectively, based on gastrointestinal symptoms and severity of pneumonia; and the peak levels of AST (28 vs 48 vs 109, P < 0.001) and ALT (33 vs 47.5 vs 106, P = 0.0114) were significantly stratified according to these criteria[13].

Apart from the liver enzyme tests mentioned above, there are characteristic clinical manifestations of liver damage. In China, it has been reported that some patients recovering from severe COVID-19 exhibited darkening and pigmentation during the recovery process. Multiple organ damage, especially liver damage, is the main cause of darkening and hyperpigmentation[14].

Abnormal liver function may lead to pigmentation through the following three pathways:

  • (1) Impaired liver function leads to hypofunction of the adrenal cortex. When the liver is unable to metabolize the melanin-stimulating hormone secreted by the anterior pituitary gland, the secretion of melanin increases[15];
  • (2) abnormal liver function hinders the inactivation of estrogen, leading to an increase in its level. The increase in estrogen levels in the body reduces the inhibition of tyrosinase by thiamine, thereby increasing the conversion of tyrosine to melanin[16];
  • (3) liver damage increases the iron content in the blood. Iron delivered to the facial skin causes the darkening of the face.

Liver biopsy is also important in the etiological diagnosis of hepatic injury in COVID-19, particularly in cases where liver damage dominates the clinical manifestation, or where other alternative causes of damage need to be ruled out.

Currently, most of the information on histological changes in the liver of patients with COVID-19 comes from autopsies. A case series by Bradley et al[17], based on ultrastructural findings and histopathology of samples from 14 fatal COVID-19 infections in the Washington State, showed centrilobular necrosis, consistent with hypoperfusion injury, in four patients; viral RNA was detected in the liver of these patients, as well.

However, autopsies have several limitations. Death may occur long after the acute liver injury is noted; subsequently, histological changes may have been eliminated or obscured, and viral load would have diminished over time. Fiel et al[18] presented their findings from two liver biopsies performed on patients infected with SARS-CoV-2.

Both patients had severe hepatic failure in the absence of obvious involvement of other organs. Detailed histological analysis, in situ hybridization, and electron microscopy revealed that apoptosis, abundant mitosis, mixed inflammatory infiltration in the portal area, severe bile duct injury, apparent viral particles, and viral RNA within hepatocytes are typical.

These findings suggested hepatic involvement in infections with SARS-CoV-2. Another case report by Melquist et al[19] showed similar findings in a patient infected with SARS-CoV-2, manifesting as acute hepatitis without any respiratory symptoms, rapidly progressing to fulminant liver failure. Acute hepatitis (panacinar hepatitis, zone 3 necrosis, and focal hemophagocytosis) with viral-like changes was identified at the time of liver biopsy.

POTENTIAL MECHANISMS OF LIVER INJURY IN PATIENTS WITH COVID-19

The available evidence supports that hepatic injury in SARS-CoV-2 infection is a consequence of a multifactorial attack. The potential mechanisms of pathogenesis may be broad spectrum, ranging from direct cytotoxicity from viral infection to indirect involvement of the inflammatory cytokine storm, hypoxic changes caused by respiratory failure, endotheliitis, and drug-induced liver injury (DILI)[20] (Figure 1).

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Figure 1
Underlying mechanisms of coronavirus disease-19-assocaited liver injury. A: Systematic inflammatory storm, which is generated by abnormal activation of the immune system; B: Drug-induced liver injury; C: Endotheliitis and coagulation dysfunction; D: Direct cytotoxicity from severe acute respiratory syndrome coronavirus 2. SARS-CoV-2: Severe acute respiratory syndrome coronavirus 2.

Direct effect of viral infection on the liver

Recently, it was determined by quantitative reverse transcription-polymerase chain reaction that SARS-CoV-2 RNA is widely present in other organs outside the respiratory tract, such as the liver[21], although the exact cell location of replication has not been determined because of the isolation of nucleic acids by whole tissue homogenization.

Until recently, a typical hepatitis picture is yet to be observed, and hepatic tropism and direct cytopathic effects of SARS-CoV-2 should be considered as the underlying mechanism of COVID-19 associated liver injury[22]. A major determinant of viral tropism is the availability of viral receptors on the surface of host cells in specific tissues.

Cellular entry of SARS-CoV-2 is mediated by the spike (S) protein of the virus, which is cleaved by transmembrane serine protease 2/transmembrane serine protease 4 and specifically interacts with angiotensin converting enzyme 2 (ACE2) in the host[23] (Figure ​2).

According to Human Protein Atlas, ACE2 is highly expressed in the lung (type II alveolar cells), intestine, and gall bladder, but it seems to be almost absent in the liver. After in-depth research on ACE2 expression patterns, sinusoidal endothelial cells appear to be negative for ACE2, but this protein is expressed in the central hepatic vein and portal vein endothelial cells[24].

The expression level of ACE2 in the bile duct epithelium is comparable to that in alveolar epithelial cells, being almost 20-times higher than that in hepatocytes. Of note, Letko et al[25] revealed that compensatory differentiation and proliferation of liver parenchymal cells derived from bile duct cells leads to the upregulation of ACE2 expression in liver tissues, which might be the underlying mechanisms in COVID-19-associated liver injury.

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Figure 2
Proposed structure diagram of severe acute respiratory syndrome coronavirus 2 and its life cycle in host cells. A: Structural sketch of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2); B: Recognition and entry of SARS-CoV-2 into host cell. Transmembrane spike (S) glycoprotein on the surface of SARS-CoV-2 forms a homotrimer to recognize the human host angiotensin converting enzyme 2 (ACE2) receptor. The S protein is specifically cleaved by two mucose-specific serine proteases [recombinant transmembrane protease serine 2 (TMPRSS2) and TMPRSS4] and furine. The subunit of S protein (S1) is released, and another subunit (S2) is exposed and mediates the viral entry into host cells; C: Life cycle of SARS-CoV-2 in host cells. First, the S protein of SARS-CoV-2 binds to ACE2 to form an S protein-ACE2 complex, which directly mediates the cellular entry of virus and the process is facilitated by TMPRSS2, TMPRSS4, and furine. Second, viral RNA is released into host cytoplasm. Open reading frame (ORF) 1a and ORF1ab are translated into large polyproteins by host ribosome, which are further proteolytically cleaved into 16 non-structural proteins (nsps). Viral polymerase protein is assembled by nsps and viral replication/transcription complex (vRTC) is subsequnently formed by polymerase protein and genomic RNA. Third, a negative sense viral RNA is synthesized and used as a template to replicate progeny (+) sense viral genome and transcribes to form various mRNAs. The nucleocapsid protein is translated in the cytoplasm, whereas the S protein, membrane (M) protein, and envelope (E) protein are translated in the endoplasmic reticulum and transported to the Golgi apparatus for further packaging. Finally, a completely new viral particle is assembled by viral RNA-nucleocapsid complex and S, M, and E proteins in endoplasmic reticulum–Golgi intermediate compartment and is released from host cell via exocytosis. SARS-CoV-2: Severe acute respiratory syndrome coronavirus 2; M: Membrane; E: Envelope; S: Spike; ER: Endoplasmic reticulum; vRTC: Viral replication/transcription complex; ORF: Open reading frame; TMPRSS: Transmembrane protease serine; ACE2: Angiotensin converting enzyme 2.

Notably, typical coronavirus particles (characterized by S structures) were identified in the cytoplasm of hepatocytes in an ultrastructural examination by Wang et al[26]. In this study, typical lesions of viral infection, including conspicuous mitochondrial swelling, decreased glycogen granules, and endoplasmic reticulum dilatation, were also observed in the SARS-CoV-2-infected hepatocytes, which indicated that hepatic impairment might be directly caused by SARS-CoV-2.

Interestingly, massive hepatic apoptosis and some binuclear hepatocytes were also identified in this study. In addition, autopsy results in patients with SARS, in the study by Guo et al[27], showed a large number of hepatocyte balloons, central lobular necrosis, and obvious apoptosis.

Similar histological findings were also observed in a study of liver biopsy from patients with SARS by Chau et al[28], which suggested that SARS-CoV may induce apoptosis of hepatocytes, thereby leading to liver damage. Furthermore, Tan et al[29] demonstrated that overexpression of p7a, a protein specifically expressed in SARS-CoV-infected cells, could induce apoptosis in cell lines derived from different organs (including lung, kidney, and liver) via a caspase-dependent pathway. This further confirmed the possibility that SARS-CoV can directly attack liver tissues and cause liver damage.

It is noteworthy that the expression level of ACE2 on hepatocytes is regulated by many factors. Several experimental studies, in both mice and humans, have confirmed increased expression of hepatic ACE2 under conditions of liver fibrosis/cirrhosis[30]. This may partially explain why pre-existing CLD increases the probability of liver damage in patients with COVID-19.

Hypoxia, a typical feature in severe COVID-19 cases, has been proven to be a main regulator of ACE2 expression in hepatic cells[31]. This may explain why the dissemination of SARS-CoV-2 outside the lungs is mainly observed in patients with acute respiratory distress syndrome and other hypoxic conditions. Notably, the affinity of S protein in SARS-CoV-2 for its receptor can be increased when it is proteolytically activated by trypsin, a protein commonly expressed in liver epithelial cells[32].

A clinical drug trial by Fantini et al[33] indicated that ganglioside (GM1) might be another target that influences the S protein–ACE2 interaction, using a combination of structural and molecular modeling approaches. In the near future, new molecular and therapeutic insights concerning the S protein–ACE2 interactor are expected to be uncovered with the advancement of research.

Inflammatory storm in COVID-19-associated hepatic injury

Inflammatory cytokine storm generated by the excessive immune response induced by coronavirus infection might also be one of the key factors in hepatic injury[34,35]. Higher plasma levels of inflammatory cytokines (IL-2, IL-7, IL-10, GSCF, IP10, MCP1, MIP1A, and TNF-α) and lower lymphocyte counts (both helper T cells and suppressor T cells) were commonly observed in patients with COVID-19, especially in the critically ill ones[4,36]. Qin et al[37] showed that COVID-19 is a systemic inflammatory viral response, first during the viral infection period and subsequently during the inflammatory period.

This may explain why the conditions of illness in some patients with COVID-19 are not serious in the early stage, but if they do not receive timely medical treatment, the disease deteriorates rapidly in a short time and enters a state of multiple organ failure[38].

A cohort study of 192 patients revealed that an increase in IL-6 and IL-10 and a decrease in CD4+ T cells were independent risk factors related to severe liver damage[39]. In another study recently published in World J Gastroenterol[40], lymphopenia and C-reactive protein levels were found to be independently associated with hepatic injury (Figure ​3).

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Figure 3
Underlying molecular mechanisms of coronavirus disease-19-associated liver injury caused by systematic inflammatory response syndrome and hypoxic ischemia. (a): Complement and interleukin-23 are released into the blood during the systemic inflammation, which subsequently activate Kupffer cells and induce their production of tumor necrosis factor α (TNF-α). As a pro-inflammatory cytokine, TNF-α aggravates the inflammation responses by up-regulating the expression of endothelial cell adhesion molecules and inducing hepatocytes to secrete chemokines. Under the induction of chemokines, CD4 T cells and neutrophils are rapidly recruited to the liver, in which CD4 T cells assist mucosal molecules to promote neutrophils into the liver parenchyma. Finally, neutrophils directly damage liver cells by releasing oxidants and proteases, leading to necrotic cell death; (b): Acute respiratory distress syndrome and endotheliitis are the two main causes leading to hypoxic-ischemic liver injury in the period of systematic inflammatory response syndrome. Increased anaerobic glycolysis leads to a decrease in ATP production, which ultimately leads to the death of hepatocytes by inhibiting hepatocyte signal transduction. ARDS: Acute respiratory distress syndrome; TNF-α: Tumor necrosis factor α; IL: Interleukin; IFN-γ: Interferon-γ.

Burra[14] confirmed that the incidence of liver damage in patients with COVID-19 having elevated ferritin levels was significantly higher (52.3% vs 20.0%) than that of patients with normal ferritin levels. This suggests that ferritin could be employed as an easy-to-use tool to ascertain liver injury.

A possible reason for this is that ferritin, acting as an inflammatory cytokine like IL-6, participates in acute liver damage[41]. Inflammasome activation and apoptosis/pyrolysis in SARS-CoV-2 induced inflammatory cells may cause multiorgan dysfunction[42]. Interestingly, pathological changes, such as spleen atrophy and lymph node necrosis, were observed in severe cases of SARS infection, which indicated the presence of immune-mediated injury[43].

Endotheliitis in COVID-19-associated hepatic injury

COVID-19 is considered to be a thrombo-inflammatory disease that affects the lungs and, beyond that, endothelium, which is one of the largest organs in the human body. A variety of viruses, such as the HIV, dengue fever virus, and Ebola virus, have been previously reported to affect the coagulation system[44].

SARS-CoV-2 enters the endothelial cells by endocytosis via binding to the ACE2 receptor as well[45]. A recent study from Switzerland[42] showed the presence of viral inclusion structures within endothelial cells and diffuse endothelial inflammation. The vascular endothelium is indispensable in regulating the vascular tone and in maintaining vascular homeostasis, and intact endothelial cells provide potent anti-coagulant properties[46].

When the vascular endothelium is destroyed, either directly by viral infection or through immune-mediated inflammation, vasoconstriction and procoagulant behavior can occur rapidly. Spiezia et al[47] found that plasma levels of fibrinogen and D-dimer in severe cases of COVID-19 were significantly higher than those in healthy controls. In this study, markedly hypercoagulable thromboelastometry profiles, as reflected by shorter clot formation time and higher maximum clot firmness, were also observed in patients with COVID-19.

In a recent study, multiple areas of microthrombi were revealed in a patient with COVID-19 by contrast-enhanced ultrasound of the lung[48], which confirmed the involvement of microvessels during the disease process. In addition, the frequency of acute pulmonary embolus in patients with COVID-19 was 30%[49] higher than that usually occurring in critically ill (1.3%)[50] or emergency (3% to 10%)[51] patients without COVID-19 (Figure ​3).

SARS-CoV-2 infection causes inflammation of the vascular endothelium, which in turn leads to vascular dysfunction, especially in capillaries. Subsequently, microvascular dysfunction leads to a hypercoagulable state, tissue edema, and organ ischemia[44,52]. Liver ischemia reperfusion injury, a pathophysiological process commonly occurring after rapid recovery of blood circulation, may be the underlying mechanism of COVID-19-associated hepatic injury.

Liver ischemia-reperfusion can activate neutrophils, Kupffer cells, and platelets, inducing a series of destructive cellular reactions, such as reactive oxygen species and calcium overload, which ultimately lead to an inflammatory response and cell damage. It has also been reported that hepatic sinusoidal endothelial cell damage causes microcirculation disorders and further aggravates liver ischemia and hypoxia. Wang et al[9] observed different degrees of hypoxemia by blood gas analysis in more than 40% of patients with COVID-19.

Patients receiving oxygen therapy have a faster recovery of liver function, and the average length of hospital stay is considerably shortened. In addition, lymphatic vessels are also reported to be involved in the pathological process of acute liver injury, prevent the occurrence of acute liver damage, and delay the progression of COVID-19[53]. Lymphatic vessels participate in the clearance of virus through absorption and transportation of inflammatory exudates, inflammatory cytokines, dead cell debris, and immune cells[54].

reference link : https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8192279/


Autoimmune hepatitis after COVID-19 vaccine – more than a coincidence

There have been some concerns regarding the possibility of COVID-19 vaccine-induced autoimmunity [1]. Molecular mimicry was suggested as a potential mechanism for this association. Indeed, antibodies against the spike protein S1 of SARS-CoV-2 had a high affinity against some human tissue proteins [2]. As vaccine mRNA codes the same viral protein, they can trigger autoimmune diseases in predisposed patients.

We report the case of a 65-year-old woman, with JAK2 V617F-positive polycythemia vera diagnosed in 2006, under pegylated interferon since 2019. Her routine medication also included acetylsalicylic acid 100 mg/day, sertraline 25 mg/day and esomeprazole 20 mg/day for over two years. She had no history of liver disease and was known to have normal routine liver tests (AST 28U/L, ALT 24U/L, GGT 24U/L, ALP 108U/L, total bilirubin 0.72 mg/dL). Moreover, she had no personal or family history of autoimmune disease.

After receiving the first dose of Moderna-COVID-19 vaccine, the patient presented mild abdominal pain. Two weeks later, routine liver function tests showed AST 1056U/L, ALT 1092U/L, GGT 329U/L, ALP 24U/L, total bilirubin 1.14 mg/dL. Complete blood cell count and international normalized ratio were normal. She denied recent changes in drug therapy. The serology for hepatitis A virus, human immunodeficiency virus, cytomegalovirus, Epstein-Barr virus and herpes simplex virus type 1 and 2 were all negative. Polymerase chain reaction for hepatitis B, C and E viruses were also negative. Ceruloplasmin, alpha-1 antitrypsin and iron tests were normal, as well as thyroid function. Antinuclear antibody was positive (1:100, speckled pattern), detected by indirect immunofluorescence assay on HEP-20-10 cells/monkey liver (initial dilution 1/100; final dilution 1/1000). Anti-mitochondrial, anti-smooth muscle, anti-liver-kidney microsomal, anti-soluble liver antigen and antineutrophil cytoplasmic antibodies were all negative. At this point, serum IgA, IgM and IgG levels were normal. Abdominal Doppler ultrasound showed hepatomegaly without cirrhotic morphology, and no biliary dilation or thrombosis.

Five weeks after vaccination, the patient presented with jaundice and choluria. Liver profile was worsening and IgG levels were now elevated (Fig. 1). The patient was admitted for clinical management. A percutaneous liver biopsy was performed, revealing a marked expansion of the portal tracts due to dense inflammatory infiltrate, with aggregates of plasma cells; severe interface hepatitis and multiple confluent foci of lobular necrosis were also observed (Fig. 2).

Fig. 1
Fig. 1. – Evolution of liver function tests (A), total bilirubin (B) and total IgG levels (C) over time. AST – aspartate aminotransferase, ALT – alanine aminotransferase, GGT – gamma-glutamyl transferase, ALP – alkaline phosphatase, IgG – Immunoglobulin G.
Fig. 2
Fig. 2. Liver biopsy findings – (A) Marked portal tract inflammation with intense lymphoplasmacytic infiltrate and interface hepatitis (HE, 30x). The inflammation consists primarily of lymphocytes and aggregates of plasma cells, with few eosinophils. (B) Intense lobular activity associated with centrilobular necrosis (HE 20x).

The score of simplified diagnostic criteria of the International Autoimmune Hepatitis Group was 8 [definite diagnosis of autoimmune hepatitis (AIH)]. Treatment with prednisolone 60 mg/day was started with a quick improvement of liver function tests and normalization of IgG levels. One month after initial diagnosis, the patient remains well on a tapering course of corticosteroids.

Recently, some cases of AIH that developed after COVID-19 vaccination have been reported [3,4]. There are some similarities between the previously described cases and the present case, such as a short interval between vaccination and symptoms onset [3]. Although our patient received the Moderna mRNA vaccine, there are already other reports of AIH induced by the Pfizer-BioNTech and Oxford-AstraZeneca vaccines, supporting the idea that the COVID-19 vaccine triggers autoimmune phenomena regardless of its mechanism of action [4].

In contrast to other cases, our patient did not have confounding factors such as pregnancy or autoimmune conditions [3,4]. However, she was under treatment with pegylated interferon for polycythemia vera. In fact, this drug has been linked to induction of autoimmune conditions, including AIH [5]. Nevertheless, this side effect usually arises within 1–2 months of starting therapy and our patient had already started pegylated interferon for over two years. In addition, the timing of the liver injury, the extensive exclusion of other causes of hepatic disease and the normalization of liver function tests and IgG levels after treatment, make it very likely that AIH was triggered by COVID-19 vaccination.

Only long-term follow-up will confirm whether COVID-19 vaccination increases the risk of AIH. Nevertheless, it should not distract healthcare providers from the overwhelming benefits of mass COVID-19 vaccination.

reference link : https://www.sciencedirect.com/science/article/pii/S0896841121001499

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